US 20200148771A1 INI (19 ) United States (12 ) Patent Application Publication ( 10) Pub . No .: US 2020/0148771 A1 BAEUERLE et al. (43 ) Pub . Date : May 14 , 2020

( 54 ) INDUCIBLE MONOVALENT ANTIGEN Publication Classification BINDING PROTEIN (51 ) Int. Ci. ( 71 ) Applicant: Harpoon Therapeutics , Inc. , South CO7K 16/28 (2006.01 ) (52 ) U.S. CI. San Francisco , CA (US ) CPC CO7K 16/2818 ( 2013.01 ) ; CO7K 16/2878 (72 ) Inventors : Patrick BAEUERLE , South San (2013.01 ) ; CO7K 2317/526 ( 2013.01 ) ; CO7K Francisco , CA (US ) ; Holger WESCHE , 2317/524 (2013.01 ) ; CO7K 2317/75 ( 2013.01 ) ; South San Francisco , CA (US ) CO7K 2317/565 ( 2013.01) ; CO7K 2317/35 (2013.01 ) ; CO7K 2317/622 (2013.01 ) ; COOK ( 21 ) Appl. No.: 16 /489,523 2317/92 ( 2013.01 ); CO7K 2319/50 (2013.01 ) ( 22 ) PCT Filed : Feb. 28 , 2018 (57 ) ABSTRACT ( 86 ) PCT No .: PCT /US2018 / 020307 Disclosed herein are inducible monovalent target -binding $ 371 ( c ) ( 1 ) , proteins which are activated upon protease cleavage . Phar (2 ) Date : Aug. 28 , 2019 maceutical compositions comprising the binding proteins Related U.S. Application Data disclosed herein and methods of using such formulations are ( 60 ) Provisional application No. 62/ 465,000 , filed on Feb. further provided . 28, 2017 . Specification includes a Sequence Listing .

scFv1 SCFVZ Anti - Target binding domain Secondpolypeptide Firstpolypeptide CH2 - Fc1 CH2 -Fc2 CH2 - Fc1 CH2 - Fc2 CH3 - Fc1 B CH3 -Fc2 Proteolytic cleavage CH3 - Fc1 CH3 - Fc2 heterodimer FC1 - Fc2 heterodimer Inactive Active Patent Application Publication May 14 , 2020 Sheet 1 of 5 US 2020/0148771 A1

Fig. 1

schyi scFv2 Anti- Target binding domain polypeptideSecond VHH VI Firstpolypeptide CH2 -Fc1 CH2 -Fc2 CH2 - Fc1 CH2 - Fc2 CH3 - Fc1 CH3 - FcZ Proteolytic cleavage CH3 - Fc1 CH3- Fc2 heterodimer FC1 - FC2 heterodimer Inactive Active Patent Application Publication May 14 , 2020 Sheet 2 of 5 US 2020/0148771 A1

Fig . 2

Inert VL

Protezse - clesvable Protease

FC prwis

Prodrug Active Drug Patent Application Publication May 14 , 2020 Sheet 3 of 5 US 2020/0148771 A1

Fig . 3

Anti - CD40 Anti- CTLA4 ProMonoMabs ProMonoMabs

Dace Seli Treme Antibody

*** + ?? option 32 motions Protease

66 Patent Application Publication May 14 , 2020 Sheet 4 of 5 US 2020/0148771 A1

SetProtocowab(activated

ma Selictelumab(ant-CD40) Coronkacw Tremetiena(anti-CTLA4} Concentration

3 ) A450 ( Binding CTLA4 4.Fig ) 4450( Binding C040

kockets-15 ma 1 WE Dacenzurabfani-C040) ???? 38 Cmontsion Ipilimutsb(BILA4 CurvedSize am

) A450 ( Binding GOO ) A460 ( Brxding OTLA4 2888 Patent Application Publication May 14 , 2020 Sheet 5 of 5 US 2020/0148771 A1

-SelProfvionoMabactivated()

1000 100 Crosslinked Concentration(nu

) OD640( Signaling CD40

5.Fig

SeliProMano-Mabactivated(,

10 Concentration(nka) Non-crosslinked ma

38 0.2 ) OD640( Signaling CD40 US 2020/0148771 A1 May 14 , 2020

INDUCIBLE MONOVALENT ANTIGEN [ 0010 ] In some embodiments , the iVL comprises a BINDING PROTEIN CDRL1, CDRL2 , and CDRL3 , and the at least one protease cleavage site of the iVL is located within the CDRL1, CROSS -REFERENCE CDRL2 , or CDRL3 . In some embodiments , the iVH com prises a CDRH1, CDRH2, and CDRH3, and the at least one [0001 ] This application claims the benefit of U.S. Provi protease cleavage site of the iVH is located within the sional Application No. 62/ 465,000 , filed Feb. 28, 2017 , CDRH1, CDRH2, or CDRH3. In some embodiments , the which is incorporated by reference herein in its entirety . first polypeptide chain comprises a further protease cleavage site , and wherein the second polypeptide comprises a further INCORPORATION BY REFERENCE protease cleavage site . In some embodiments , the further [ 0002 ] All publications, patents , and patent applications protease cleavage site of the first polypeptide chain is mentioned in this specification are herein incorporated by located within the Ll or the Ll' , and the further protease reference to the same extent as if each individual publica cleavage site of the second polypeptide chain is located tion , patent, or patent application was specifically and indi within the L6 or the L6 '. In some embodiments , the Fc1 and vidually indicated to be incorporated by reference , and as if Fc2 each independently comprise one or more amino acid set forth in their entireties . substitutions favoring formation of a heterodimeric Fc region . In some embodiments , the CH3 domain of the Fc1 BACKGROUND OF THE INVENTION comprises a substitution of an amino acid with a negatively [0003 ] The present disclosure provides an inducible mon charged amino acid , and the CH3 domain of the Fc2 ovalent target -binding protein which are activated upon comprises a substitution of an amino acid with a positively protease cleavage, and can be used for diagnosing and charged amino acid . In some embodiments , the CH3 domain treating cancers. of the Fcl comprises a substitution of an amino acid with a positively charged amino acid , and the CH3 domain of the SUMMARY OF THE INVENTION Fc2 comprises a substitution of an amino acid with a [0004 ] In one embodiment is provided , an inducible mon negatively charged amino acid . In some embodiments , the ovalent target - binding protein , wherein the protein com iVH and the iVL each independently comprise one or more prises a first polypeptide chain and a second polypeptide mutations that prohibit target binding of said domains . In chain , some embodiments , the VH , iVL , and the L1 or the Ll' of [0005 ] the first polypeptide chain comprising : a VH the first polypeptide form a scFv. In some embodiments , the target- binding domain (VH ) , an inactive VL domain VL , IVH , and the L6 or the L6 ' of the second polypeptide ( VL ) that binds to the VH domain , a linker (L1 ) , and form a scFv. a first monomeric Fc domain (Fcl ) comprising a CH3 [0011 ] In one embodiment is provided , an inducible mon and a CH2 domain ; ovalent target - binding protein , wherein the protein com [0006 ] the second polypeptide chain comprising : a VL prises a first scFv ( scFv1 ) and a second scFv ( scFv2 ) , and a target- binding domain (VL ) , an inactive VH domain first (Fc1 ) and a second ( Fc2 ) monomeric Fc domain , each ( VH ) that binds to the VL domain , a linker ( L6 ) , a comprising a CH2 domain and a CH3 domain , second monomeric Fc domain (Fc2 ) comprising a CH3 [0012 ] the scFv1 comprising : a VH target- binding and a CH2 domain ; domain (VH ) , an inactive VL domain ( VL ) that binds wherein the iVL and the iVH each comprise at least one to the VH domain , and a (L1 ) ; protease cleavage site , wherein upon activation by protease [0013 ] the scFv2 comprising: a VL target -binding cleavage of the at least one protease cleavage site of both the domain ( VL ), an inactive VH domain (iVH ) thatbinds iVL and the iVH , the VH and the VL associate to form an to the VL domain , and a linker (L6 ) ; active target- binding domain , and wherein the CH3 domains wherein the scFv1 and the scFv2 each comprise at least one of the Fcl and Fc2 form a heterodimer . protease cleavage site , wherein upon activation by protease [0007 ] In one embodiment is provided , An inducible mon cleavage of the protease cleavage sites, the VH and the VL ovalent target - binding protein , wherein the protein com associate to form an active target -binding domain , and prises a first polypeptide chain and a second polypeptide wherein the Fcl and the Fc2 form a Fc region comprising a chain , heterodimeric CH3 domain . [0008 ] the first polypeptide chain comprising : a VL target- binding domain (VL ) , an inactive VH domain [0014 ] In one embodiment is provided , an inducible mon (iVH ) that binds to the VL domain , a linker (L1 ' ) , and ovalent target -binding protein , wherein the protein com a first monomeric Fc domain (Fc1 ) comprising a CH3 prises a first scFv (scFvl ) and a second scFv (scFv2 ), and a and a CH2 domain ; first (Fcl ) and a second ( Fc2 ) monomeric Fc domain , each [ 0009 ] the second polypeptide chain comprising: a VH comprising a CH2 domain and a CH3 domain , target- binding domain (VH ) , an inactive VL domain [ 0015 ] the scFv1 comprising: a VL target -binding ( VL ) that binds to the VH domain , a linker (L6 ') , a domain (VL ), an inactive VH domain ( iVH ) that binds second monomeric Fc domain (Fc2 ) comprising a CH3 to the VL domain , and a linker (L1 ' ) ; and a CH2 domain ; [0016 ] the scFv2 comprising: a VH target- binding wherein the iVH and the iVL each comprise at least one domain (VH ) , an inactive VL domain (AVL ) that binds protease cleavage site , wherein upon activation by protease to the VH domain , and a linker (L6 ') ; cleavage of the at least one protease cleavage site of both wherein the scFv1 and the scFv2 each comprise at least one iVH and the iVL , the VL and the VH associate to form an protease cleavage site , wherein upon activation by protease active target- binding domain , and wherein the CH3 domains cleavage of the at least one protease cleavage site of both the of the Fcl and the Fc2 form a heterodimer. scFv1 and the scFv2 , the VL and the VH associate to form US 2020/0148771 A1 May 14 , 2020 2 an active target -binding domain , and wherein the Fcl and [0018 ] One embodiment provides an inducible monova the Fc2 form a Fc region comprising a heterodimeric CH3 lent target -binding protein , wherein the protein comprises a domain . first polypeptide chain and a second polypeptide chain , [ 0019 ] the first polypeptide chain comprising : a VH [ 0017 ] In some embodiments , the iVL comprises a target- binding domain ( VH ) , an inactive VL domain CDRL1, CDRL2 , and CDRL3 , and the at least one protease ( VL ) that binds to the VH domain , a linker (L1 ) , and cleavage site of the iVL is located within the CDRLI , a first monomeric Fc domain (Fc1 ) comprising a CH3 CDRL2 , or CDRL3. In some embodiments , the iVH com and a CH2 domain ; prises a CDRH1, CDRH2, and CDRH3, and the at least one [ 0020 ] the second polypeptide chain comprising : a VL protease cleavage site of the iVH is located within the target- binding domain ( VL ) , an inactive VH domain CDRH1, CDRH2, or CDRH3. In some embodiments , the ( VH ) that binds to the VL domain , a linker (L6 ), a scFv1 comprises a further protease cleavage site , and second monomeric Fc domain (Fc2 ) comprising a CH3 wherein the scFv2 comprises a further protease cleavage and a CH2 domain ; site . In some embodiments , the further protease cleavage site wherein the L1 and L6 each comprise at least one protease of the scFv1 is located within the Ll or the Ll' , and the cleavage site , wherein upon activation by protease cleavage further protease cleavage site of the scFv2 is located within of the at least one protease cleavage site ofboth L1 and L6 , L6 or L6 '. In some embodiments , the Fcl and the Fc2 each the VH and the VL associate to form an active target -binding independently comprise one or more amino acid substitution domain , and wherein the CH3 domains of the Fcl and Fc2 in their respective CH3 domain favoring formation of the Fc form a heterodimer. region comprising a heterodimeric CH3 domain . In some [ 0021 ] One embodiment provides an inducible monova embodiments , the CH3 domain of the Fcl comprises a lent target- binding protein , wherein the protein comprises a substitution of an amino acid with a negatively charged first polypeptide chain and a second polypeptide chain , amino acid , and the CH3 domain of the Fc2 comprises a [0022 ] the first polypeptide chain comprising : a VL substitution of an amino acid with a positively charged target- binding domain ( VL ), an inactive VH domain amino acid . In some embodiments , the CH3 domain of the ( VH ) that binds to the VL domain , a linker (L1 ' ) , and Fel comprises a substitution of an amino acid with a a first monomeric Fc domain (Fc1 ) comprising a CH3 positively charged amino acid , and the CH3 domain of the and a CH2 domain ; Fc2 comprises a substitution of an amino acid with a [0023 ] the second polypeptide chain comprising : a VH negatively charged amino acid . In some embodiments , the target- binding domain (VH ), an inactive VL domain iVH and the iVL domains each independently comprise one (iVL ) that binds to the VH domain , a linker (L6 ') , a or more mutation that prohibit target binding of said second monomeric Fc domain (Fc2 ) comprising a CH3 domains . In some embodiments , the active target -binding and CH2 domain ; domain binds to a target expressed on a tumor cell . In some wherein the Ll' and L6 ' each comprise at least one protease embodiments , the active target -binding domain binds to a cleavage site , wherein upon activation by protease cleavage tumor antigen . In some embodiments , the active target of the at least one protease cleavage site of both Ll' and L6 ', binding domain binds to an immune checkpoint protein . In the VL and the VH associate to form an active target- binding some embodiments, the active target -binding domain binds domain , and wherein the CH3 domains of the Fcl and Fc2 to CD27 , CD40 , OX40 , GITR , CD137 , B7, CD28 , ICOS , form a heterodimer. A2AR , B7 -H3 , B7 -H4 , BTLA , CTLA - 4 , IDO , KIR , LAG3, [ 0024 ] In some embodiments , the inducible monovalent PD - 1, PD - L1 , TIM - 3 , VISTA . In some embodiments , the at target- binding protein of claim 29 or 30 , wherein the iVL least one protease cleavage site is recognized by at least one comprises a CDRLI , CDRL2 , and CDRL3 . In some of a serine protease, a cysteine protease , an aspartate pro embodiments , the iVH comprises a CDRH1, CDRH2 , and tease , a threonine protease , a glutamic acid protease , a CDRH3 . In some embodiments , the Fcl and the Fc2 each metalloproteinase , a gelatinase, and a asparagine peptide independently comprise one or more amino acid substitu lyase . In some embodiments , the at least one protease tions favoring formation of a heterodimeric Fc region . In cleavage site is recognized by at least one of a Cathepsin B , some embodiments , the CH3 domain of the Fcl comprises a Cathepsin C , a Cathepsin D , a Cathepsin E , a Cathepsin K , a substitution of an amino acid with a negatively charged a Cathepsin L , a kallikrein , a hk1, a hK10 , a hk15 , a amino acid , and the CH3 domain of the Fc2 comprises a plasmin , a collagenase , a Type IV collagenase , a stromely substitution of an amino acid with a positively charged sin , a Factor Xa, a chymotrypsin -like protease, a trypsin - like amino acid . In some embodiments , the CH3 domain of the protease , a elastase - like protease, a subtilisin - like protease , Fcl comprises a substitution of an amino acid with a an actinidain , a bromelain , a calpain , a caspase , a caspase - 3 , positively charged amino acid , and the CH3 domain of the a Mir1 -CP , a papain , a HIV - 1 protease , a HSV protease , a Fc2 comprises a substitution of an amino acid with a CMV protease , a chymosin , a renin , a pepsin , a matriptase , negatively charged amino acid . In some embodiments , the a legumain , a plasmepsin , a nepenthesin , a metalloexopep iVH and the iVL each independently comprise one ormore tidase , a metalloendopeptidase , a matrix metalloprotease mutations that prohibit target binding of said domains. In (MMP ), a MMP1, a MMP2, a MMP3, a MMP8 , a MMP9 , some embodiments , the VH , the iVL , and the L1 or the Ll' a MMP10 , a MMP11 , a MMP12 , a MMP13 , a MMP14 , an of the first polypeptide form a scFv. In some embodiments , ADAM10 , an ADAM12, an urokinase plasminogen activa the iVH , and the L6 or the L6 ' of the second polypeptide tor ( UPA ), an enterokinase , a prostate - specific target (PSA , form a scFv . hK3) , an interleukin - 1ß converting enzyme, a thrombin , a [0025 ] One embodiment provides an inducible monova FAP (FAP - a ), a dipeptidyl peptidase , and a dipeptidyl lent target- binding protein , wherein the protein comprises a peptidase IV (DPPIV /CD26 ) . first scFv ( scFvl ) and a second scFv ( scFv2 ) , and a first US 2020/0148771 A1 May 14 , 2020 3

(Fcl ) and a second ( Fc2 ) monomeric Fc domain , each aspartate protease , a threonine protease , a glutamic acid comprising a CH2 domain and a CH3 domain , protease , a metalloproteinase , a gelatinase , and a asparagine [0026 ] the scFv1 comprising : a VH target- binding peptide lyase . domain (VH ), an inactive VL domain ( iVL ) that binds to the VH domain , and a (L1 ) ; BRIEF DESCRIPTION OF THE DRAWINGS [0027 ] the scFv2 comprising : a VL target- binding [0032 ] The novel features of the invention are set forth domain (VL ), an inactive VH domain (iVH ) that binds with particularity in the appended claims. A better under to the VL domain , and a linker (L6 ) ; standing of the features and advantages of the present wherein the scFvl and the scFv2 each comprise at least one invention will be obtained by reference to the following protease cleavage site , wherein upon activation by protease detailed description that sets forth illustrative embodiments , cleavage of the protease cleavage sites , the VH and the VL in which the principles of the invention are utilized, and the associate to form an active target -binding domain , and accompanying drawings of which . wherein Fcl and Fc2 form a Fc region comprising a het [0033 ] FIG . 1 shows an embodiment of the inducible erodimeric CH3 domain . monovalent target -binding protein of the present disclosure [ 0028 ] One embodiment provides an inducible monova illustrating activation of the target -binding protein upon lent target- binding protein , wherein the protein comprises a proteolytic cleavage of at least one protease cleavage site in first scFv ( scFvl ) and a second scFv (scFv2 ) , and a first the inactive VL domain and at least one protease cleavage ( Fcl) and a second (Fc2 ) monomeric Fc domain , each site in the inactive VH domain . comprising a CH2 domain and a CH3 domain , [0034 ] FIG . 2 shows an inducible monovalent antibody [0029 ] the scFvl comprising: a VL target- binding according to the present disclosure illustrating activation of domain (VL ), an inactive VH domain ( iVH ) that binds the antibody , from its prodrug from to the active drug form , to the VL domain , and a linker ( L1' ) ; upon proteolytic cleavage of at least one protease cleavage [0030 ] the scFv2 comprising: a VH target- binding site in the linker between the VL and the inactive VH domain domain (VH ) , an inactive VL domain (iVL ) that binds of one polypeptide chain and the linker between the VH and to the VH domain , and a linker (L6 ') ; inactive VL domain of another polypeptide chain . wherein the scFv1 and the scFv2 each comprise at least one [ 0035 ] FIG . 3 shows proteolytic activation of four exem protease cleavage site , wherein upon activation by protease plary inducible monovalent antibodies of this disclosure , cleavage of the at least one protease cleavage site of both the Lanes 1 and 2 correspond to Dace -ProMono Mab , Lanes 3 scFv1 and the scFv2 , the VL and the VH associate to form and 4 correspond to Seli- ProMonoMab , Lanes 5 and 6 an active target - binding domain , and wherein the Fc1 and correspond to Ipi- Pro Mono Mab , and Lanes 7 and 8 corre Fc2 form a Fc region comprising a heterodimeric CH3 spond to Treme- ProMonoMab . The lanes marked as “ _ ” domain . correspond to unactivated ProMono Mab and the lanes [ 0031] In some embodiments , the iVL comprises a marked as “ + ” correspond to proteolytically activated CDRL1, CDRL2 , and CDRL3 . In some embodiments , the ProMonoMab . The indicated antibodies denote parental iVH comprises a CDRH1, CDRH2 , and CDRH3 . In some antibodies from which the VH and VL sequences were embodiments , the at least one protease cleavage site of the derived . Dace , dacetuzumab ; seli, selicrelumab ; ipi, ipilim scFv1 is located within the Ll or the Ll'. In some embodi umabv ; treme, tremelimumab . ments , the further protease cleavage site of the scFv2 is [0036 ] FIG . 4 shows target binding of four exemplary located within the L6 or the L6 '. In some embodiments , the inducible monovalent antibodies of this disclosure . Top left Fel and the Fc2 each independently comprise one or more panel shows CD40 binding of Dace -ProMonoMab in prod amino acid substitution in their respective CH3 domain rug (dotted line ) and activated (solid line ) forms; top right favoring formation of the Fc region comprising a heterodi panel shows CD40 binding of Seli -ProMono Mab in prodrug meric CH3 domain . In some embodiments , the CH3 domain (dotted line ) and activated ( solid line ) forms; bottom left of the Fcl comprises a substitution of an amino acid with a panel shows CTLA4 binding of Ipi- Pro MonoMab in prod negatively charged amino acid , and the CH3 domain of the rug (dotted line ) and activated ( solid line ) forms; and bottom Fc2 comprises a substitution of an amino acid with a right panel shows CTLA4 binding of Treme- ProMonoMab positively charged amino acid . In some embodiments , the in prodrug (dotted line ) and activated ( solid line ) forms. CH3 domain of the Fcl comprises a substitution of an amino [0037 ] FIG . 5 shows CD40 binding of Seli -Pro Mono Mab acid with a positively charged amino acid , and the CH3 upon protease activation . This assay uses HEK293 cells that domain of the Fc2 comprises a substitution of an amino acid are engineered to express CD40 and are also engineered to with a negatively charged amino acid . In some embodi express and secrete alkaline phosphatase when CD40 is ments , the iVH and the iVL domains each independently activated . The amount of secreted alkaline phosphatase is comprise one or more mutation that prohibit target binding measure using a substrate that changes color proportionate of said domains . In some embodiments , the active target to the amount of secreted alkaline phosphatase . Left panel binding domain binds to a target expressed on a tumor cell . shows CD40 activation in cells by Seli -ProMono Mab in In some embodiments , the active target - binding domain prodrug (dotted line ) and activated ( solid line ) forms. Right binds to a tumor antigen . In some embodiments , the e active panel shows CD40 activation in cells by Seli -ProMono Mab target -binding domain binds to an immune checkpoint pro in prodrug (dotted line ) and activated ( solid line ) forms, tein . In some embodiments , the active target - binding domain crosslinked with anti -human IgG Fc polyclonal antibody . binds to CD27 , CD40 , OX40 , GITR , CD137 , B7 , CD28 , ICOS, A2AR , B7- H3 , B7 -H4 , BTLA , CTLA - 4 , IDO , KIR , DETAILED DESCRIPTION OF THE LAG3, PD - 1 , PD - L1, TIM - 3 , VISTA . In some embodi INVENTION ments , the at least one protease cleavage site is recognized [0038 ] While preferred embodiments of the present inven by at least one of a serine protease , a cysteine protease , an tion have been shown and described herein , it will be US 2020/0148771 A1 May 14 , 2020 4

obvious to those skilled in the art that such embodiments are Immunological Interest, Fifth Edition , National Institute of provided by way of example only . Numerous variations, Health , Bethesda , Md. ( 1991 ) ) . The constant domains are changes, and substitutions will now occur to those skilled in not involved directly in binding an antibody to an antigen , the art without departing from the invention . It should be but exhibit various effector functions , such as participation understood that various alternatives to the embodiments of of the antibody in antibody - dependent cellular toxicity . the invention described herein may be employed in practic “ Variable domain residue numbering as in Kabat” or “ amino ing the invention . It is intended that the following claims acid position numbering as in Kabat ,” and variations thereof, define the scope of the invention and that methods and refers to the numbering system used for heavy chain variable structures within the scope of these claims and their equiva domains or light chain variable domains of the compilation lents be covered thereby of antibodies in Kabat et al. , Sequences of Proteins of Certain Definitions Immunological Interest, 5th Ed . Public Health Service , National Institutes of Health , Bethesda, Md. ( 1991 ). Using [0039 ] The terminology used herein is for the purpose of this numbering system , the actual linear amino acid describing particular cases only and is not intended to be sequence may contain fewer or additional amino acids limiting . As used herein , the singular forms “ a ” , “ an ” and “ the” are intended to include the plural forms as well , unless corresponding to a shortening of, or insertion into , a FR or the context clearly indicates otherwise . Furthermore , to the CDR of the variable domain . For example, a heavy chain extent that the terms “ including ” , “ includes ” , “ having” , variable domain may include a single amino acid insert “ has” , “ with ” , or variants thereof are used in either the ( residue 52a according to Kabat) after residue 52 ofH2 and detailed description and / or the claims, such terms are inserted residues ( , residues 82a , 82b , and 82c , etc according intended to be inclusive in a manner similar to the term to Kabat ) after heavy chain FR residue 82. The Kabat " comprising ." numbering of residues may be determined for a given [0040 ] The term “ about” or “ approximately ” means within antibody by alignment at regions of homology of the an acceptable error range for the particular value as deter sequence of the antibody with a “ standard ” Kabat numbered mined by one of ordinary skill in the art, which will depend sequence . It is not intended that CDRs of the present in part on how the value is measured or determined , the disclosure necessarily correspond to the Kabat numbering limitations of the measurement system . For example , convention . " about" can mean within 1 or more than 1 standard devia [ 0044 ] A " single chain Fv ” or “ scFv ” , as used herein , tion , per the practice in the given value . Where particular refers to a binding protein in which the variable domains of values are described in the application and claims, unless the heavy chain and of the light chain of a traditional two otherwise stated the term “ about” should be assumed to chain antibody are joined to form one chain . Typically , a mean an acceptable error range for the particular value. linker peptide is inserted between the two chains to allow for [0041 ] The terms “ individual ,” “ patient, ” or “ subject ” are proper folding and creation of an active binding site . used interchangeably . None of the terms require or are limited to situation characterized by the supervision ( e.g. [ 0045 ] A “ cleavage site for a protease, " as meant herein , is constant or intermittent) of a health care worker ( a doctor , a an amino acid sequence that can be cleaved by a protease , registered nurse , a nurse practitioner, a physician's assistant, such as, for example , a matrix metalloproteinase or a furin . an orderly , or a hospice worker) . Examples of such sites include Gly -Pro - Leu -Gly - Ile - Ala [0042 ] The term “ Framework ” or “ FR ” residues (or Gly -Gln or Ala - Val- Arg - Trp - Leu -Leu - Thr- Ala , which can regions ) refer to va ble main residues other than the be cleaved by metalloproteinases, and Arg - Arg -Arg - Arg CDR or hypervariable region residues as herein defined . A Arg - Arg , which is cleaved by a furin . In therapeutic appli “ human consensus framework ” is a framework which rep cations, the protease cleavage site can be cleaved by a resents the most commonly occurring amino acid residue in protease that is produced by target cells, for example cancer a selection of human immunoglobulin VL or VH framework cells or infected cells , or pathogens. sequences . [ 0046 ] As used herein , " elimination half - time” is used in [0043 ] As used herein , “ Variable region ” or “ variable its ordinary sense , as is described in Goodman and Gill domain ” refers to the fact that certain portions of the variable man's The Pharmaceutical Basis of Therapeutics 21-25 domains differ extensively in sequence among antibodies (Alfred Goodman Gilman , Louis S. Goodman , and Alfred and are used in the binding and specificity of each particular Gilman , eds. , 6th ed . 1980 ) . Briefly , the term is meant to antibody for its particular antigen . However, the variability encompass a quantitative measure of the time course of drug is not evenly distributed throughout the variable domains of elimination . The elimination of most drugs is exponential antibodies. It is concentrated in three segments called ( i.e., follows first- order kinetics) , since drug concentrations complementarity -determining regions ( CDRs ) or hypervari usually do not approach those required for saturation of the able regions both in the light- chain and the heavy -chain elimination process . The rate of an exponential process may variable domains. The more highly conserved portions of be expressed by its rate constant, k , which expresses the variable domains are called the framework (FR ). The vari fractional change per unit of time, or by its half - time, t1/ 2 the able domains of native heavy and light chains each comprise time required for 50 % completion of the process. The units four FR regions, largely adopting a B -sheet configuration , of these two constants are time- and time, respectively . A connected by three CDRs, which form loops connecting , and first- order rate constant and the half- time of the reaction are in some cases forming part of, the Bsheet structure . The simply related (kxt1 / 2 = 0.693) and may be interchanged CDRs in each chain are held together in close proximity by accordingly . Since first- order elimination kinetics dictates the FR regions and , with the CDRs from the other chain , that a constant fraction of drug is lost per unit time, a plot contribute to the formation of the antigen -binding site of of the log of drug concentration versus time is linear at all antibodies ( see Kabat et al. , Sequences of Proteins of times following the initial distribution phase ( i.e. after drug US 2020/0148771 A1 May 14 , 2020 5

absorption and distribution are complete ). The half- time for DARPins, fynomers , kunitz domain peptides , and mono drug elimination can be accurately determined from such a bodies, a ligand or peptide . In some embodiments , the graph . inactive domain is an inactive variable heavy ( VH ) or an [0047 ] Described herein are inducible monovalent target inactive variable light ( iVL ) domain . The inducible protein binding proteins, pharmaceutical compositions as well as is in a binding inactive configuration when the inhibitory or inactive domain is associated with at least one other domain nucleic acids, recombinant expression vectors , and host cells of the protein , in either the first or the second polypeptide of for making such inducible monovalent binding proteins. the inducible monovalent target -binding protein , such that Also provided are methods of using the disclosed inducible said binding domain is prevented from binding its target . In monovalent binding proteins in the prevention , and / or treat its binding inactive configuration neither the first polypep ment of diseases, conditions and disorders. The inducible tide nor the second polypeptide is able to bind a target. monovalent target - binding proteins of this disclosure are [0049 ] The at least two inactive domains each comprises heteromultimeric proteins having a binding inactive form at least one protease cleavage site . The protease cleavage and an active form . In the binding inactive form , the protein sites are a stretch of amino acid sequences that are recog has an immunoglobulin like structure with two arms, nized and cleaved by any known protease , such as matrix whereas in the active form the two arms associate with each metalloprotease (, MMP9 ) , or furin . In some cases , an other to form a monovalent structure , as shown in FIGS . 1 inactive domain comprising a protease cleavage site recog and 2. The inducible monovalent target- binding proteins are nized by MMP9 comprises the amino acid sequence PR (S / activated by cleavage of protease sites located within at least T ) ( L / I) ( S / T ) (SEQ ID NO . 3 ) . In some cases, an inactive two of the domains of the heteromultimeric structure. In domain comprising a protease cleavage site recognized by some embodiments, the inducible monovalent binding pro MMP9 comprises the amino acid sequence LEATA (SEQ ID teins are capable of specifically binding to a tumor antigen . NO : 4 ) . In some cases, the protease cleavage site is recog In some embodiments , the inducible monovalent target nized in a sequence - specific manner by a MMP11. In some binding proteins are half -life extended through an Fc cases , the protease cleavage site recognized by a MMP11 domain , and accordingly has a prolonged elimination half comprises a polypeptide having an amino acid sequence life . In some instance , the inducible monovalent target GGAANLVRGG (SEQ IN NO : 5 ) . In some cases, the binding proteins are heteromultimeric antibodies. In some protease cleavage site is recognized by a protease disclosed cases when a normal or healthy tissue expresses the same in Table 1. In some cases, the protease cleavage site recog antigens as a tumor cell, toxicities are possible due to nized by a protease disclosed in Table 1 comprises a off- tumor antigen binding . In some embodiments , the induc polypeptide having an amino acid sequence selected from a ible monovalent target -binding proteins disclosed herein are sequence disclosed in Table 1 (SEQ ID NOS : 1-42 ) . advantageously activated in a tumor tissue specific manner , [0050 ] Proteases are proteins that cleave proteins, in some upon exposure to proteases prevalent in tumor microenvi cases, in a sequence - specific manner. Proteases include but ronment. The inhibitory domains obstruct the binding are not limited to serine proteases , cysteine proteases , aspar domains and accordingly prevent binding of the proteins to tate proteases, threonine proteases , glutamic acid proteases , their targets until the inhibitory domains are cleaved by metalloproteases, asparagine peptide lyases , serum pro proteases . teases, cathepsins, Cathepsin B , Cathepsin C , Cathepsin D , Cathepsin E , Cathepsin K , Cathepsin L , kallikreins, hK1, Inactive Domains hK10 , hK15 , plasmin , collagenase , Type IV collagenase , [0048 ] In one aspect , the disclosure provides an inducible stromelysin , Factor Xa , chymotrypsin - like protease, trypsin monovalent target- binding protein comprising at least two like protease , elastase - like protease, subtilisin - like protease , inactive domains. In some embodiments , the inactive actinidain , bromelain , calpain , caspases , caspase -3 , Mirl domains comprise protease cleavage sites . The inactive CP, papain , HIV - 1 protease , HSV protease , CMV protease , domains are alternatively referred to as inhibitory domains chymosin , renin , pepsin , matriptase, legumain , plasmepsin , or inert domains . Examples of inactive domains include but nepenthesin , metalloexopeptidases , metalloendopeptidases, are not limited to a variable heavy domain (VH ) , a variable matrix metalloproteases (MMP ), MMP1 , MMP2 , MMP3, light domain (VL ) , an scFv comprising a VH and a VL MMP8 , MMP9, MMPP13 , MMP11 , MMPP14 , urokinase domain , a single domain antibody, or a variable domain of plasminogen activator (UPA ) , enterokinase , prostate -specific camelid derived nanobody (VHH ), a non - Ig binding domain , antigen (PSA , hK3) , interleukin -1B converting enzyme, i.e. , antibody mimetic , such as anticalins , affilins, affibody thrombin , FAP (FAP - a ), dipeptidyl peptidase , and dipepti molecules , affimers , affitins, alphabodies , avimers, dyl peptidase IV ( DPPIV / CD26 ) . TABLE 1 Exemplary Proteases and Protease Recognition Sequences Cleavage Domain Protease Sequence SEQ ID NO :

MMP7 KRALGLPG 1

MMP 7 ( DE ) GRPLALWRS ( DR ) 8 2 MMP9 PR ( S / T ) ( L / I ) ( S / T ) 3

MMP9 LEATA 4 US 2020/0148771 A1 May 14 , 2020 6

TABLE 1 - continued Exemplary Proteases and Protease Recognition Sequences Cleavage Domain Protease Sequence SEQ ID NO : MMP11 GGAANLVRGG 5

MMP14 SGRIGFLRTA

MMP PLGLAG 7

MMP PLGLAX 8 MMP PLGC (me ) AG 9

MMP ESPAYYTA 10 MMP RLQLKL 11 MMP RLQLKAC 12 MMP2 , MMP9 , MMP14 EP ( Cit ) G (Hof ) YL 13 Urokinase plasminogen activator (UPA ) SGRSA 14 Urokinase plasminogen activator ( UPA ) DAFK 15 Urokinase plasminogen activator ( UPA ) GGGRR 16 Lysosomal Enzyme GFLG 17

Lysosomal Enzyme ALAL 18 Lysosomal Enzyme FK 19

Cathepsin B NLL 20

Cathepsin D PIC (Et ) FF 21 Cathepsin K GGPRGLPG 22 Prostate Specific Antigen HSSKLO 23 Prostate Specific Antigen HSSKLQL 24 Prostate Specific Antigen HSSKLQEDA 25 Herpes Simplex Virus Protease LVLASSSFGY 26 HIV Protease GVSQNYPIVG 27 CMV Protease GVVQASCRLA 28 Thrombin F (Pip ) RS 29

Thrombin DPRSFL 30

Thrombin PPRSFL 31

Caspase - 3 DEVD 32 Caspase - 3 DEVDP 33

Caspase - 3 KGSGDVEG ?? Interleukin 1ß converting enzyme GWEHDG 35 Enterokinase EDDDDKA 36 FAP KQEQNPGST 37

Kallikrein 2 GKAFRR 38

Plasmin DAFK 39

Plasmin DVLK 40 US 2020/0148771 A1 May 14 , 2020 7

TABLE 1 - continued Exemplary Proteases and Protease Recognition Sequences Cleavage Domain Protease Sequence SEQ ID NO : Plasmin DAFK 41

TOP ALLLALL 42

[0051 ] Proteases are known to be secreted by some dis domain , wherein the Fc domain comprises a CH3 and a CH2 eased cells and tissues , for example tumor or cancer cells , domain . CH2 and CH3 domain molecules are small in size , creating a microenvironment that is rich in proteases or a usually less than 15 kD . It is contemplated that in certain protease - rich microenvironment . In some case , the blood of embodiments the CH2 and CH3 domain molecules are a subject is rich in proteases . In some cases , cells surround conjugated to an effector molecule or a label. In other ing the tumor secrete proteases into the tumor microenvi embodiments , CDRs/ hypervariable amino acid sequences ronment. Cells surrounding the tumor secreting proteases are inserted into the CH3 and / or CH2 domain . In such include but are not limited to the tumor stromal cells , embodiments , the CH2 or CH3 domain vary in size depend myofibroblasts , blood cells , mast cells , B cells , NK cells , ing on the length of CDR /hypervariable amino acid regulatory T cells , macrophages, cytotoxic T lymphocytes , sequence inserted in the loops regions, how many CDRs are dendritic cells ,mesenchymal stem cells , polymorphonuclear inserted and whether another molecule ( such as an effector cells, and other cells . In some cases, proteases are present in molecule or label) is conjugated to the CH2 or CH3 domain . the blood of a subject, for example proteases that target In some embodiments , the Fc domain does not comprise amino acid sequences found in microbial peptides. This additional constant domains ( i.e. CH1) . In one embodiment, feature allows for targeted therapeutics such as antigen the CH3 and / or CH2 domain is from IgG , IgA or IgD . In binding proteins to have additional specificity because T another embodiment , the CH3 and / or CH2 domain is from cells will not be bound by the antigen binding protein except IgE or IgM . CH2 and CH3 domain molecules can be in the protease rich microenvironment of the targeted cells glycosylated or unglycosylated . For example, a recombinant or tissue. CH2 or CH3 domain can be expressed in an appropriate mammalian cell to allow glycosylation of the molecule . Inducible Monovalent Target -Binding Proteins [0055 ] In some embodiments , the inducible monovalent target -binding protein disclosed herein is an Fc domain [0052 ] In some embodiments , the inducible monovalent molecule or an Fc fusion protein . In some embodiments , the target -binding proteins described herein comprise a first and inducible monovalent target -binding domains described a second polypeptide chain , each polypeptide comprising an herein comprise a first and second monomeric Fc domains , inactive variable domain , a variable heavy domain ( VH ) or Fcl and Fc2 , respectively , which monomeric Fc domains a variable light domain (VL ) , and an Fc region . The inactive associate with each other to form a heterodimeric Fc domain is a variable light domain ( iVL ) or a variable heavy domain , and wherein a first scFv ( scFvl ) is attached to the domain ( iVH ) ; and the Fc region comprises a CH3 domain N -terminus of Fc1, and a second scFv ( scFv2 ) is attached to with amino acid substitutions , and a CH2 domain . In some the N -terminus of Fc2 , and further wherein the scFv1 and embodiments , the inactive domains iVL and iVH each scFv2 each comprise at least one protease cleavage site . The comprise at least one protease cleavage site which is cleaved first monomeric Fcl domain comprises two domains CH3 by a protease to result in activation of the inducible mon Fel and CH3- Fc2 , and the second monomeric Fc2 domain ovalent target - binding proteins described herein . comprises two domains CH3 - Fc2 and CH2 - Fc2. Each scFv1 [0053 ] In some embodiments , the first polypeptide com and scFv2 comprises an inactive domain , and a variable light prises an iVL , a VH , and a first Fc domain (Fc1 ) comprising (VL ) or variable heavy domain (VH ). In some embodiments , CH3- Fc1 and CH2- Fc1 ; the second polypeptide comprises the inactive domains are iVL and iVH , and each comprises an iVH , a VL , and a second Fc domain ( Fc2 ) comprising at least one protease cleavage site which is cleaved by a CH3 -Fc2 and CH2- Fc2 , which VH and VL associate to form protease to result in activation of the inducible monovalent the target -binding domain of the inducible monovalent pro target- binding proteins described in this embodiment. The tein , and wherein CH3 - Fcl and CH3 - Fc2 form a heterodi protease cleavage sites of the inactive domains are , in some mer , as shown in FIG . 1. In some embodiments , the first cases, within the complementarity determining regions of polypeptide comprises an iVH , a VL , and a first Fc domain those domains ; or within the linkers that connects the (Fc1 ) comprising CH3- Fcl and CH2- Fc1 ; the second poly inactive domains iVL and iVH with the VH or VL domains, peptide comprises an iVL , a VH , and a second Fc domain respectively . ( Fc2 ) comprising CH3- Fc2 and CH2- Fc2 , which VL and VH [0056 ] In some embodiments , scFvl comprises an iVH associate to form the target- binding domain of the inducible domain and a VL domain . In some embodiments , scFv1 monovalent protein , and wherein CH3- Fcl and CH3 - Fc2 comprises an iVL domain and a VH domain . In some form a heterodimer. embodiments , scFv2 comprises an iVH domain and a VL [0054 ] An Fc domain conjugated to an effector molecule domain . In some embodiments , scFv2 comprises an iVL is referred to as an Fc domain molecule or an Fc domain domain and a VH domain . In some embodiments , scFv1 fusion protein . The fusion protein can include, for example , comprises an iVL domain and a VH domain ; scFv2 com a VH , a engineered antibody domain , a diabody, an scFv, a prises an iVH domain and a VL domain , wherein upon cytokine, a toxin , an enzyme, and /or a ligand attached an Fc cleavage of the at least one protease cleavage site in iVH and US 2020/0148771 A1 May 14 , 2020 8 iVL the VL and VH domains associate to form an active L6 ' have different lengths . In certain embodiments , internal monovalent target - binding protein . In some embodiments , linkers L1 , L2 , L3 , and /or L4 are “ short” , i.e., consist of 0 , scFv1 comprises an iVH domain and a VL domain ; scFv2 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 or 12 amino acid residues . Thus, comprises an iVL domain and a VH domain , wherein upon in certain instances , the linkers consist of about 12 or less cleavage of the at least one protease cleavage site in iVH and amino acid residues . In the case of O amino acid residues, the iVL , the VH and VL domains associate to form an active linker is a peptide bond . In certain embodiments , linkers L1, monovalent target -binding protein . L1 , L2 , L2 , L3, L3' , L4 , L4 ', L5 , L5 ', L6 , and L6 ' consist [ 0057 ] The inactive variable domains iVL and iVH each of 15 , 20 or 25 amino acid residues. In some embodiments , comprises complementarity determining regions CDRLI, the linkers consist of about 3 to about 15 , for example 8 , 9 CDRL2 , CDRL3 , and CDRH1, CDRH2 , CDRH3, respec or 10 contiguous amino acid residues . Regarding the amino tively , and the at least one protease cleavage site is , in certain acid composition of the linkers L1 , L1' , L2 , L2 , L3 , L3 , L4 , embodiments , located within said complementarity deter L4 ', L5 , L5 ', L6 , and L6 ', peptides are selected with prop mining regions . It is contemplated that, in some embodi erties that confer flexibility to the antigen - binding proteins, ments , the CDRs of iVL and /or iVH further comprise one or do not interfere with the target - binding domain as well as more mutations that prohibit the binding of said domains to resist cleavage from proteases, unless the protease cleavage a target . sites are located within the linkers . Examples of internal [ 0058 ] The proteins described herein are binding inactive linkers suitable for linking the domains in the antigen in the two - armed form , and only bind target protein when in binding proteins include but are not limited to (GS ) n (GGS ) the monovalent form . In some embodiments , the proteins n ? (GGGS ) n (GGSG )n . (GGSGG )n , or (GGGGS ) n , wherein described herein do not have target- domain binding capa nis 1 , 2 , 3 , 4 , 5,6,7,8,9 , or 10. In one embodiment, internal bility until at least one protease cleavage site in iVL and at linker L1, L1' , L2 , L2 , L3 , L3 , L4 , L4 ', L5 , L5 ', L6 , and /or least one cleavage site in iVH are cleaved by a protease and L6 ' is (GGGGS ), or (GGGGS ) 3. the VH and VL domains associate with each other to form [0061 ] In certain instances, one or more of the linkers L1 , a monovalent active target -binding domain . In some L1' , L2 , L2 , L3, L3 , L4, L4 ', L5 , L5' , L6 , and L6 ' comprise embodiments , the proteins do not have target - domain bind protease cleavage sites. Such protease sensitive linkers are , ing capability until all the protease cleavage sites in iVH and in certain embodiments , sensitive to protease present in iVL are cleaved and the VH and VL domains associate with specific tissue or intracellular compartments ( , MMPs, furin , each other to form a monovalent active target -binding cathepsin B ). Example sequences for such protease sensitive domain . cleavable linkers include but are not limited to (PLGLWA )n , [0059 ] The VH and iVL domains, the VL and iVH (RVLAEA ) .: ( EDVVCCSMSY ) , (GGIEGRGS ) , which domains , the VL and CH2 domain , the VH and CH2 domain , are recognized by MMP -1 , and (GFLG ), which are recog and the CH3 and CH2 domains are connected to each other nized by furin . The linkers containing protease cleavage by internal linkers. In embodiments where the first polypep sites play a role in activation of the inducible monovalent tide comprises an iVL , a VH , and a first Fc domain (Fc1 ) target -domain binding protein . In some embodiments , the comprising CH3- Fcl and CH2 -Fc1 ; the second polypeptide binding protein is activated upon cleavage of the protease comprises an iVH , a VL , and a second Fc domain ( Fc2 ) sites in iVL , iVH , and one or more of linkers L1 , L1 , L2 , comprising CH3 -Fc2 and CH2 -Fc2 , the linkers are as fol L2 , L3 , L3 , L4 , L4' , L5 , L5' , L6 , and L6 '. In embodiments lows : L1 links iVL and VH of the first polypeptide ; L2 links where one or more of the linkers comprise protease cleavage VH and CH2 - Fc1 ; L3 links CH2- Fcl and CH3 - Fc1 ; L4 links sites the inducible monovalent target -domain binding pro CH3 - Fc2 and CH2 -Fc2 ; L5a links CH2- Fc2 and VL ; and L6 tein is not activated until at least one cleavage site in at least links VL and iVH of the second polypeptide. In embodi one of the linkers is cleaved . ments where the first polypeptide comprises an iVH , a VL , [0062 ] It is contemplated that in some embodiments the and a first Fc domain (Fcl ) comprising CH3 - Fcl and inducible monovalent target -binding protein is no more than CH2- Fcl ; the second polypeptide comprises an iVL , a VH , 100 kD , no more than 75 kD , no more than 50 kD , no more and a second Fc domain (Fc2 ) comprising CH3- Fc2 and than 25 kD , no more than 20 kD , no more than 15 kD , or no CH2 - Fc2 , the linkers are as follows: Ll' links iVH and VL more than 10 kD , upon its activation by protease cleavage . of the first polypeptide ; L2 ' links VH and CH2- Fc1 ; L3' links Prior to cleavage and activation , the inducible protein is , in CH2- Fc1 and CH3 - Fc1 ; L4 ' links CH3- Fc2 and CH2- Fc2 ; certain embodiments , no more than 100 kD , no more than 75 L5' links CH2 -Fc2 and VL ; and L6 ' links VH and IVL of the kD , no more than 50 kD , no more than 25 kD , no more than second polypeptide. In some embodiments, the linkers L1, 20 kD , or no more than 15 kD . Ll' , L6 , and L6 ', each independently comprises at least one protease cleavage site which is cleaved by a protease to Fc - Domain Heterodimerization result in activation of the inducible monovalent target [ 0063] Although the wild - type Fc region is a homodimer binding proteins described herein of polypeptides , the Fc domains disclosed herein comprise [ 0060] Linkers L1 , L1' , L2 , L2 , L3 , L3 , L4 , L4 ', L5 , L5 ', amino acid substitutions such that they do not form homodi L6 , and L6 ' have an optimized length and /or amino acid mers. The monomeric Fc domains, Fc1 and Fc2 , are in some composition . In some embodiments , linkers L1 , L1' , L2 , L2' , embodiments IgG Fc. In some embodiments , themonomeric L3 , L3 , L4 , L4 ', L5 , L5 ', L6 , and L6 ' are 3-200 amino acids Fc domains, Fcl and Fc2, are from other immunoglobulin in length . In some embodiments , linkers L1 , L1' , L2 , L2 , L3 , subclasses including IgA , IgE , IgD , and IgM . L3 , L4 , L4 ', L5, L5 ', L6 , and L6 ' have the same length or [0064 ] The heterodimer Fc region of the inducible mon amino acid composition . In other embodiments, linkers L1, ovalent target -binding proteins described herein comprises a L1 , L2 , L2' , L3, L3 , L4 , L4' , L5 , L5' , L6 , and L6 ' have variant CH3 constant domain comprising amino acid muta different amino acid compositions. In other embodiments , tions that promote the formation of said heterodimer with linkers L1 , L1' , L2 , L2 , L3 , L3 ', L4 , L4' , L5 , L5' , L6 , and stability comparable to a native homodimeric Fc , and a CH2 US 2020/0148771 A1 May 14 , 2020 9 constant domain . The wild -type Fc is homodimeric in nature with the displayed CH3- Fcl or CH3A . Fluorescence -based and this feature is driven by both hydrophobic interactions detection of this interaction enables screening of the library at the center of the CH3 interface and symmetric electro for heterodimeric Fc variants by flow cytometry . static interactions around the rim of the hydrophobic core. In [0066 ] The Y - shape of typical IgG antibodies often limits some embodiments , the Fc domain described herein com their utility against some targets , whereby bivalent target prises amino acid substitutions such that they do not form binding dimerizes and agonizes , rather than antagonizes, the homodimers. In some embodiments , the Fc domain intended targets (See , Choi et al. , Engineering of Immuno described herein comprises amino acid substitutions that globulin Fc heterodimers using yeast surface -displayed favor formation of heterodimers over homodimers . In some combinatorial Fc library screening , PLOS One, 10 ( 12 ): embodiments , the variant Fc domain is created using ( i ) e0145349 , 2015. Therefore , in some embodiments, the symmetric - to -asymmetric steric complementarity design (, inducible monovalent target -binding proteins described KiH , HA - TF, and ZW1) , ( ii ) charge -to -charge swap ( , DD herein are advantageously used for antagonization of bind KK ), ( iii ) charge - to - steric complementarity swap plus addi ing targets . In some embodiments , the heterodimerization of tional long - range electrostatic interactions (, EW - RVT ) , and the Fel and Fc2 domains is expected to reduce high molecu ( iv ) isotype strand swap [ , strand - exchange engineered lar weight tetrameric by - products during preparation of the domain (SEED ) ]. Strand exchange mutations include, for inducible monovalent target -domain binding proteins dis example , IgA -derived 45 residues on IgG1 CH3- Fcl and closed herein . IgG1- derived 57 residues on IgA CH3 - Fc2 , or vice versa . [ 0067] An antibody that includes a wild -type Fc domain Examples of symmetric - to -asymmetric sterically comple has the ability to interact with neonatal Fc- receptor (FcRn ) mentary mutations include HA - TF (S364H / F405A in Fc1 in a pH dependent manner; this interaction confers extended CH3 or CH3A and Y349T / T394F in Fc2 - CH3 or CH3B ) , serum half -life . The residues important for the high -affinity ZW1( T350V / L351Y /F405A / Y407V in Fc1 -CH3 or CH3A interaction of Fc domain and FcyR are located within the and T350V / T366L / K392L / T394W in Fc2 - CH3 or CH3B ) . CH2 domain . Accordingly , in some embodiments , CH2 - Fc1 In some embodiments , the Fc variant is generated using the and CH2 -Fc2 comprise wild type IgG sequence. “ Knobs - into - holes (KiH )” approach where Fcl comprises a T366W “ knob " mutation , in Fc1 - CH3 or CH3A , and Fc2 Activated Monovalent Target- Binding Protein comprises T366S /L368A /Y407V " hole " mutations in Fc2 [0068 ] The inducible monovalent target- binding proteins CH3 or CH3B domain . In some embodiments , the Fc variant described herein are activated by cleavage of the at least one is generated using the “ Knobs - into -holes (KiH ) ” plus dis protease cleavage site in the inactive VL domain , and the at ulfide bond approach , KiHs-s , where Fcl comprises a least one protease cleavage site in the inactive VH domain . T366W /S354C “ knob " mutation , in Fc1 - CH3 or CH3A , and Upon protease cleavage the VH and VL target binding Fc2 comprises T366S /L368A / Y407V / Y349C “ hole ” muta domains associate to form a monovalent active target tions in Fc2 -CH3 or CH3B domain . In such exemplary binding domain . It is contemplated that the target antigen is embodiments , the heterodimerization is favored through involved in and /or associated with a disease , disorder or hydrophobic interactions at the core of the Fc1 -CH3 or condition . In particular , a target antigen associated with a CH3A and Fc2 - CH3 or CH3B interface. Examples of proliferative disease, a tumorous disease , an inflammatory charge - charge swap mutations, where the Fc heterodimer disease , an immunological disorder, an autoimmune disease , favoring interaction is based on electrostatic complementar an infectious disease , a viral disease , an allergic reaction , a ity include DD -KK (K409D /K392D in Fc1 - CH3 or CH3A parasitic reaction , a graft -versus - host disease or a host and D399K /E356K in Fc2 -CH3 or CH3B , or vice versa ). versus -graft disease . In some embodiments , a target antigens Examples of charge -to - steric complementarity swap plus is a tumor antigen expressed on a tumor cell. In some additional long -range electrostatic interaction mutations embodiments , a target antigen is a on a tumor cell, virally include EW -RVT (K360E /K409W in Fc1 -CH3 or CH3A and infected cell , bacterially infected cell , damaged red blood Q347R / D399V /F405T in Fc2 - CH3 or CH3B , or vice versa ) ; cell, arterial plaque cell, or fibrotic tissue cell. EW -RVTS - S (K360E /K409W /Y349C in Fc1 - CH3 or CH3A [0069 ] In some embodiments , a target antigen is a cell and Q347R /D399V /F405T / S354C in Fc2 - CH3 or CH3B , or surface molecule such as a protein , lipid or polysaccharide . vice versa ), which comprises an inter - CH3 S / S bond . In In some embodiments , a target antigen is an immune check some embodiments , the Fc variant is generated using hydro point protein . phobic or steric complementarity plus electrostatic comple [0070 ] Target antigens , in some cases , are expressed on the mentarity , such as 7.8.60 (K360D /D399M / Y407A in Fc1 surface of a diseased cell or tissue , for example a tumor or CH3 or CH3A and E345R / Q347R / T366V /K409V in Fc2 a cancer cell . Examples of target antigens include but are not CH3 or CH3B , or vice versa ) . limited to CD19 , CD20 , CD33 , CD30 , CD64 , CD123 , [0065 ] In certain embodiments , the heterodimer forming EpCAM , EGFR , HER - 2 , HER - 3 , c -Met , LAGU , FOIR , Fc variants described herein are generated through directed EGFR , PSMA , VEGF, and CEA . In one aspect, a target evolution combined with yeast surface display and high antigen is an immune checkpoint protein . Examples of throughput screening . For example , in some embodiments , immune checkpoint proteins include but are not limited to a combinatorial heterodimeric Fc library display system is CD27 , CD40 , OX40 , GITR , CD137 , B7 , CD28 , ICOS , developed by mating two haploid yeast cell lines ; one AZAR , B7- H3 , B7 - H4 , BTLA , CTLA - 4 , IDO , KIR , LAG3, haploid cell line displaying an Fc chain library (CH3 - Fel or PD - 1 , PD - L1 , TIM - 3 , VISTA . Inhibitory immune check CH3A ) with mutations in one CH3 domain on the yeast cell point proteins to be inhibited in activating an immune surface , and the other cell line secreting an Fc chain library response include but are not limited to A2AR , B7- H3 , (CH3 -Fc2 or CH3B ) with mutations in the other CH3 B7 -H4 , BTLA , CTLA - 4 , IDO , KIR , LAG3, PD - 1 , PD -L1 , domain . In the mated cells , secreted CH3 - Fc2 or CH3B is TIM - 3 , and VISTA . In some embodiments , binding of the displayed on the cell surface through heterodimerization inducible monovalent target- binding protein to an immune US 2020/0148771 A1 May 14 , 2020 10 checkpoint target protein is dependent upon protease cleav [0074 ] In another example of a substitution to create a age of the inhibitory domain which restricts binding of the variant inducible antibody , one or more hypervariable region protein to the immune checkpoint target protein only in the residues of a parent antibody are substituted . In general , microenvironment of a diseased cell or tissue with elevated variants are then selected based on improvements in desired levels of proteases, for example in a tumor microenviron properties compared to a parent antibody, for example , ment. increased affinity , reduced affinity , reduced immunogenicity , [0071 ] In some embodiments , the inducible monovalent increased pH dependence of binding . For example , an target- binding proteins described herein specifically binds to affinity matured variant antibody can be generated , using A2AR . In some embodiments , the inducible monovalent phage display -based affinity maturation techniques such as target -binding proteins described herein specifically binds to those described herein and known in the field . B7 -H3 . In some embodiments , the inducible monovalent [0075 ] Substitutions can be made in hypervariable regions target -binding proteins described herein specifically binds to (HVR ) of a parent inducible antibody to generate variants B7- H4 . In some embodiments , the inducible monovalent and variants are then selected based on binding affinity , i.e., target- binding proteins described herein specifically binds to by affinity maturation . In some embodiments of affinity BTLA . In some embodiments, the inducible monovalent maturation , diversity is introduced into the variable genes target- binding proteins described herein specifically binds to chosen for maturation by any of a variety of methods ( , CTLA -4 . In some embodiments , the inducible monovalent error -prone PCR , chain shuffling, or oligonucleotide -di target -binding proteins described herein specifically binds to rected mutagenesis ) . A secondary library is then created . The IDO . In some embodiments , the inducible monovalent tar library is then screened to identify any antibody variants get - binding proteins described herein specifically binds to with the desired affinity . Another method to introduce diver KIR . In some embodiments , the inducible monovalent tar sity involves HVR -directed approaches, in which several get- binding proteins described herein specifically binds to HVR residues (, 4-6 residues at a time) are randomized . LAG3 . In some embodiments , the inducible monovalent HVR residues involved in antigen binding may be specifi target -binding proteins described herein specifically binds to cally identified , e.g., using alanine scanning mutagenesis or PD -1 . In some embodiments , the inducible monovalent modeling. Substitutions can be in one , two , three , four, or target- binding proteins described herein specifically binds to more sites within a parent antibody sequence . PD -L1 . In some embodiments , the inducible monovalent [0076 ] In some embodiments , an inducible monovalent target -binding proteins described herein specifically binds to target -binding protein , as described herein comprises a VL TIM - 3 . In some embodiments , the inducible monovalent and a VH domain with amino acid sequences corresponding target- binding proteins described herein specifically binds to to the amino acid sequence of a naturally occurring VL or VISTA . VH domain , respectively , but that has been “ humanized ” , i.e., by replacing one or more amino acid residues in the Binding Protein Variants amino acid sequence of said naturally occurring VL or VH domains ( and in particular in the framework sequences ) by [0072 ] As used herein , the term “ binding protein variants ” one or more of the amino acid residues that occur at the refers to variants and derivatives of an inducible monovalent corresponding position ( s ) in a VL or VH domain from a antibody described herein . In certain embodiments , amino conventional 4 -chain antibody from a human being (, as acid sequence variants of the inducible antibodies described indicated above) . This can be performed in a manner known herein are contemplated . For example , in certain embodi in the field , which will be clear to the skilled person , for ments amino acid sequence variants of the inducible anti example on the basis of the further description herein . bodies described herein are contemplated to improve the Again , it should be noted that such humanized inducible binding affinity and / or other biological properties of the monovalent target -binding antibodies of the disclosure are antibodies . Exemplary method for preparing amino acid obtained in any suitable manner known per se and thus are variants include , but are not limited to , introducing appro not strictly limited to polypeptides that have been obtained priate modifications into the nucleotide sequence encoding using a polypeptide that comprises a naturally occurring VL the antibody, or by peptide synthesis . Such modifications and /or VH domain as a starting material . In some additional include, for example , deletions from , and /or insertions into embodiments , an inducible monovalent target -binding anti and / or substitutions of residues within the amino acid body, as described herein , comprises a VL and a VH domain sequences of the antibody. with amino acid sequences corresponding to the amino acid [0073 ] Any combination of deletion , insertion , and sub sequence of a naturally occurring VL or VH domain , respec stitution can be made to the various domains to arrive at the tively , but that has been “ camelized ”, i.e., by replacing one final construct, provided that the final construct possesses or more amino acid residues in the amino acid sequence of the desired characteristics , antigen -binding . In certain a naturally occurring VL or VH domain from a conventional embodiments , antibody variants having one or more amino 4 -chain antibody by one or more of the amino acid residues acid substitutions are provided . Sites of interest for substi that occur at the corresponding position ( s ) in a VL or a VH tution mutagenesis include the CDRs and framework domain of a heavy chain antibody. Such “ camelizing” sub regions. Amino acid substitutions may be introduced into the stitutions are preferably inserted at amino acid positions that variable domains of the target- binding protein of interest and form and /or are present at the VH - VL interface , and /or at the the products screened for a desired activity , retained /im so -called Camelidae hallmark residues (see for example WO proved antigen binding , decreased immunogenicity , or 94/04678 and Davies and Riechmann ( 1994 and 1996 )) . improved antibody -dependent cell mediated cytotoxicity Preferably , the VH sequence that is used as a starting ( ADCC ) or complement dependent cytotoxicity (CDC ) . material or starting point for generating or designing the Both conservative and non -conservative amino acid substi camelized single domain is preferably a VH sequence from tutions are contemplated for preparing the antibody variants . a mammal, more preferably the VH sequence of a human US 2020/0148771 A1 May 14 , 2020 11

being, such as a VH3 sequence. However , it should be noted Amino acid substitutions may be conservative or semi that such camelized inducible monovalent antibodies of the conservative. For example , the amino acids glycine, alanine , disclosure , in certain embodiments , are obtained in any valine , leucine and isoleucine can often be substituted for suitable manner known in the field and thus are not strictly one another ( amino acids having aliphatic side chains) . Of limited to polypeptides that have been obtained using a these possible substitutions, typically glycine and alanine polypeptide that comprises a naturally occurring VL and / or are used to substitute for one another since they have VH domain as a starting material. For example , both relatively short side chains and valine, leucine and isoleu “ humanization ” and “ camelization ” is performed by provid cine are used to substitute for one another since they have ing a nucleotide sequence that encodes a naturally occurring larger aliphatic side chains which are hydrophobic . Other VL and /or VH domain , respectively , and then changing, one amino acids which may often be substituted for one another or more codons in said nucleotide sequence in such a way include but are not limited to : phenylalanine , tyrosine and that the new nucleotide sequence encodes a “ humanized " or tryptophan (amino acids having aromatic side chains ); " camelized ” inducible antibody , respectively . This nucleic lysine, arginine and histidine (amino acids having basic side acid can then be expressed , so as to provide the desired chains) ; aspartate and glutamate ( amino acids having acidic target -antigen binding capability . Alternatively , in other side chains) ; asparagine and glutamine (amino acids having embodiments , a “ humanized ” or “ camelized ” inducible anti amide side chains) ; and cysteine and methionine (amino body is synthesized de novo using known peptide synthesis acids having sulphur -containing side chains) . In some technique from the amino acid sequence of a naturally embodiments , the inducible monovalent target -binding pro occurring antibody comprising a VL and /or VH domain . In teins are isolated by screening combinatorial libraries , for some embodiments , a “ humanized ” or “ camelized” induc example , by generating phage display libraries and screen ible antibody is synthesized de novo using known peptide ing such libraries for antibodies possessing the desired synthesis technique from the amino acid sequence or nucleo binding characteristics towards a target antigen , such as a tide sequence of a naturally occurring antibody comprising tumor antigen expressed on a cell surface . a VL and /or VH domain , respectively , a nucleotide sequence encoding the desired humanized or camelized inducible Inducible Target -Binding Protein Modifications domain antibody of the disclosure , respectively , is designed and then synthesized de novo using known techniques for [0080 ] The binding proteins described herein encompass nucleic acid synthesis , after which the nucleic acid thus derivatives or analogs in which ( i) an amino acid is substi obtained is expressed in using known expression techniques, tuted with an amino acid residue that is not one encoded by so as to provide the desired inducible antibody of the the genetic code , ( ii ) the mature polypeptide is fused with disclosure . another compound such as polyethylene glycol, or (iii ) [ 0077 ] Other suitable methods and techniques for obtain additional amino acids are fused to the protein , such as a ing the inducible antibody of the disclosure and / or nucleic leader or secretory sequence or a sequence to block an acids encoding the same, starting from naturally occurring immunogenic domain and /or for purification of the protein . sequences for VL or VH domains for example comprises [0081 ] Typical modifications include, but are not limited combining one or more parts of one or more naturally to , acetylation , acylation , ADP - ribosylation , amidation , occurring VL or VH sequences ( such as one or more covalent attachment of flavin , covalent attachment of a heme framework (FR ) sequences and / or complementarity deter moiety , covalent attachment of a nucleotide or nucleotide mining region (CDR ) sequences ) , and / or one or more syn derivative , covalent attachmentof a lipid or lipid derivative , thetic or semi- synthetic sequences , and / or a naturally occur covalent attachment of phosphatidylinositol, cross -linking , ring sequence for a CH2 domain , and a naturally occurring cyclization , disulfide bond formation , demethylation , for sequence for a CH3 domain comprising amino acid substi mation of covalent crosslinks, formation of cystine, forma tutions that favor formation of heterodimer over homodimer, tion of pyroglutamate , formylation , gamma carboxylation , in a suitable manner , so as to provide an inducible antibody glycosylation , GPI anchor formation , hydroxylation , iodi of the disclosure or a nucleotide sequence or nucleic acid nation , methylation , myristylation , oxidation , proteolytic encoding the same. processing , phosphorylation , prenylation , racemization , selenoylation , sulfation , transfer - RNA mediated addition of Affinity Maturation amino acids to proteins such as arginylation, and ubiquitina [0078 ] In designing binding proteins for therapeutic appli tion . cations, it is desirable to create proteins that , for example , [0082 ] Modifications are made anywhere in the inducible modulate a functional activity of a target , and / or improved binding proteins described herein , including the peptide binding proteins such as binding proteins with higher speci backbone , the amino acid side -chains , and the amino or ficity and / or affinity and / or and binding proteins that are carboxyl termini. Certain common peptide modifications more bioavailable , or stable or soluble in particular cellular that are useful for modification of the inducible binding or tissue environments . proteins include glycosylation , lipid attachment, sulfation , [0079 ] The inducible monovalent target- binding proteins gamma- carboxylation of glutamic acid residues, hydroxy described in the present disclosure exhibit improved the lation , blockage of the amino or carboxyl group in a poly binding affinities towards the target binding domain , for peptide, or both , by a covalent modification , and ADP example a tumor antigen expressed on a cell surface . In ribosylation . some embodiments , the inducible monovalent target -bind [0083 ] In some embodiments , the inducible monovalent ing protein of the present disclosure is affinity matured to target -binding antibodies of the disclosure are conjugated increase its binding affinity to the target binding domain , with drugs to form antibody -drug conjugates (ADCs ). In using any known technique for affinity -maturation (, muta general , ADCs are used in oncology applications, where the genesis , chain shuffling , CDR amino acid substitution ) . use of antibody - drug conjugates for the local delivery of US 2020/0148771 A1 May 14 , 2020 12 cytotoxic or cytostatic agents allows for the targeted deliv the single -substitution library encodes only a single amino ery of the drug moiety to tumors , which can allow higher acid change in its corresponding domain , or amino acid efficacy, lower toxicity , etc. segment . Typically, this allows all of the potential substitu tions in a large protein or protein binding site to be probed Polynucleotides Encoding Inducible Monovalent with a few small libraries. In some embodiments , the Target -Binding Proteins plurality of domains forms or covers a contiguous sequence [0084 ] Also provided , in some embodiments , are poly of amino acids of the inducible monovalent target binding nucleotide molecules encoding an inducible monovalent protein or a reference binding compound . Nucleotide target -binding protein as described herein . In some embodi sequences of different single -substitution libraries overlap ments , the polynucleotidemolecules are provided as a DNA with the nucleotide sequences of at least one other single construct . In other embodiments , the polynucleotide mol substitution library . In some embodiments , a plurality of ecules are provided as a messenger RNA transcript. single -substitution libraries are designed so that every mem [ 0085 ] The polynucleotide molecules are constructed by ber overlaps every member of each single - substitution known methods such as by combining the genes encoding library encoding an adjacent domain . the various domains of the inducible monovalent target [0091 ] Binding proteins expressed from such single - sub binding protein , operably linked to a suitable promoter , and stitution libraries are separately selected to obtain a subset of optionally a suitable transcription terminator , and expressing variants in each library which has properties at least as good it in bacteria or other appropriate expression system such as, as those of the reference binding compound and whose for example CHO cells . resultant library is reduced in size . Generally , the number of [0086 ] In some embodiments , the polynucleotide is nucleic acids encoding the selected set of binding com inserted into a vector, preferably an expression vector, which pounds is smaller than the number of nucleic acids encoding represents a further embodiment. This recombinant vector members of the original single - substitution library. Such can be constructed according to known methods. Vectors of properties include , but are not limited to , affinity to a target particular interest include plasmids, phagemids , phage compound , stability with respect to various conditions such derivatives, virii (e.g. , retroviruses , adenoviruses, adeno as heat, high or low pH , enzymatic degradation , cross associated viruses, herpes viruses, lentiviruses , and the like ), reactivity to other proteins and the like. The selected com and cosmids . pounds from each single -substitution library are referred to [0087 ] A variety of expression vector/ host systemsmay be herein interchangeably as “ pre -candidate compounds, " or utilized to contain and express the polynucleotide encoding " pre - candidate proteins ." Nucleic acid sequences encoding the polypeptide of the described inducible monovalent tar the pre -candidate compounds from the separate single get -binding protein . Examples of expression vectors for substitution libraries are then shuffled in a PCR to generate expression in E. coli are pSKK (Le Gall et al. , J Immunol a shuffled library , using PCR - based gene shuffling tech Methods . (2004 ) 285 ( 1 ) : 111-27 ) , pcDNA5 ( Invitrogen ) for niques . expression in mammalian cells , PICHIAPINKTM Yeast [0092 ] An exemplary work flow of the screening process Expression Systems ( Invitrogen ) , BACUVANCETM Bacu is described herein . Libraries of pre -candidate compounds lovirus Expression System (GenScript ) . are generated from single substitution libraries and selected for binding to the target protein ( s ), after which the pre [0088 ] Thus, the inducible monovalent target binding pro candidate libraries are shuffled to produce a library of teins as described herein , in some embodiments , are pro nucleic acids encoding candidate compounds which , in turn , duced by introducing a vector encoding the protein as are cloned into a convenient expression vector, such as a described above into a host cell and culturing said host cell phagemid expression system . Phage expressing candidate under conditions whereby the protein domains are compounds then undergo one ormore rounds of selection for expressed , may be isolated and, optionally , further purified . improvements in desired properties , such as binding affinity to a target molecule . Target molecules may be adsorbed or Production of Inducible Monovalent Target- Binding otherwise attached to a surface of a well or other reaction Proteins container, or target molecules may be derivatized with a [0089 ] Disclosed herein , in some embodiments , is a pro binding moiety, such as biotin , which after incubation with cess for the production of an inducible monovalent target candidate binding compounds may be captured with a binding protein of the present disclosure . In some embodi complementary moiety , such as streptavidin , bound to ments , the process comprises culturing a host transformed or beads, such as magnetic beads , for washing . In exemplary transfected with a vector comprising a nucleic acid sequence selection regimens , the candidate binding compounds encoding an inducible monovalent target binding protein undergo a wash step so that only candidate compounds with under conditions allowing the expression of the an inducible very low dissociation rates from a target molecule are monovalent target binding protein and recovering and puri selected . Exemplary wash times for such embodiments are fying the produced protein from the culture . about 10 minutes , about 15 minutes , about 20 minutes , about [0090 ] In an additional embodiment is provided a process 20 minutes , about 30 minutes, about 35 minutes , about 40 directed to improving one or more properties , affinity , sta minutes, about 45 minutes, about 50 minutes, about 55 mins , bility , heat tolerance , cross- reactivity , etc., of the inducible about 1 hour, about 2 hours , about 3 hours , about 4 hours, monovalent target binding protein described herein , com about 5 hours, about 6 hours , about 7 hours , about 8 hours ; pared to a reference binding compound . In some embodi or in other embodiments , about 24 hours ; or in other ments , a plurality of single - substitution libraries is provided embodiments , about 48 hours ; or in other embodiments , each corresponding to a different domain , or amino acid about 72 hours. Isolated clones after selection are amplified segment of the inducible monovalent target binding protein and subjected to an additional cycle of selection or analyzed , or reference binding compound such that each member of for example by sequencing and by making comparative US 2020/0148771 A1 May 14 , 2020 13 measurements of binding affinity towards their target, for individual in need thereof comprising administration of an example , by ELISA , surface plasmon resonance (SPR ) , inducible monovalent target binding protein herein . In some bio -layer interferometry (, OCTET® system , Pall Life Sci instances, the administration of the inducible monovalent ences, ForteBio , Menlo Park , Calif. ) or the like. target binding protein induces and / or sustains cytotoxicity towards a cell expressing a target antigen . In some instances, Pharmaceutical Compositions the cell expressing a target antigen is a cancer or tumor cell , [ 0093] Also provided , in some embodiments , are pharma a virally infected cell, a bacterially infected cell , an autore ceutical compositions comprising an inducible monovalent active T or B cell, damaged red blood cells , arterial plaques, target- binding protein described herein , a vector comprising or fibrotic tissue . In some embodiments , the target antigen is the polynucleotide encoding the polypeptide of the inducible an immune checkpoint protein . monovalent target binding protein or a host cell transformed [0097 ] Also provided herein are methods and uses for a by this vector and at least one pharmaceutically acceptable treatment of a disease , disorder or condition associated with carrier . The term “ pharmaceutically acceptable carrier " a target antigen comprising administering to an individual in includes , but is not limited to , any carrier that does not need thereof an inducible monovalent target binding protein interfere with the effectiveness of the biological activity of as described herein . Diseases , disorders or conditions asso the ingredients and that is not toxic to the patient to whom ciated with a target antigen include , but are not limited to , it is administered . Examples of suitable pharmaceutical viral infection , bacterial infection , auto - immune disease , carriers are well known in the art and include phosphate transplant rejection , atherosclerosis , or fibrosis . In other buffered saline solutions, water, emulsions, such as oil /water embodiments , the disease , disorder or condition associated emulsions, various types of wetting agents , sterile solutions with a target antigen is a proliferative disease , a tumorous etc. Such carriers can be formulated by conventional meth disease , an inflammatory disease, an immunological disor ods and can be administered to the subject at a suitable dose . der, an autoimmune disease , an infectious disease , a viral Preferably , the compositions are sterile . These compositions disease , an allergic reaction , a parasitic reaction , a graft may also contain adjuvants such as preservative , emulsify versus -host disease or a host -versus -graft disease . In one ing agents and dispersing agents . Prevention of the action of embodiment, the disease , disorder or condition associated microorganisms may be ensured by the inclusion of various with a target antigen is cancer. In one instance , the cancer is antibacterial and antifungal agents . a hematological cancer. In another instance , the cancer is a [0094 ] In some embodiments of the pharmaceutical com melanoma . In a further instance , the cancer is non -small cell positions, the inducible monovalent target binding protein is lung cancer. In yet further instance , the cancer is breast encapsulated in nanoparticles . In some embodiments , the cancer. In some embodiments , the inducible monovalent nanoparticles are fullerenes , liquid crystals , liposome, quan target- binding protein binds to an immune checkpoint pro tum dots , superparamagnetic nanoparticles, dendrimers , or tein and is administered to treat a cancer characterized by nanorods . In other embodiments of the pharmaceutical com upregulation of said immune checkpoint protein . For positions, the inducible monovalent target binding protein is example , the immune checkpoint protein is , in some cases , attached to liposomes . In some instances, the inducible CTLA -4 and the cancer is melanoma, non - small cell lung monovalent target binding protein is conjugated to the cancer, triple negative breast cancer, or ovarian cancer . surface of liposomes . In some instances, the inducible [0098 ] As used herein , in some embodiments, " treatment” monovalent target binding protein is encapsulated within the or “ treating ” or “ treated ” refers to therapeutic treatment shell of a liposome. In some instances , the liposome is a wherein the object is to slow (lessen ) an undesired physi cationic liposome. ological condition , disorder or disease , or to obtain benefi [ 0095 ] The inducible monovalent target binding proteins cial or desired clinical results . For the purposes described described herein are contemplated for use as a medicament. herein , beneficial or desired clinical results include , but are Administration is effected by different ways, by intravenous, not limited to , alleviation of symptoms; diminishment of the intraperitoneal, subcutaneous , intramuscular, topical or extent of the condition , disorder or disease ; stabilization intradermal administration . In some embodiments , the route ( i.e., notworsening ) of the state of the condition , disorder or of administration depends on the kind of therapy and the disease ; delay in onset or slowing of the progression of the kind of compound contained in the pharmaceutical compo condition , disorder or disease ; amelioration of the condition , sition . The dosage regimen will be determined by the disorder or disease state ; and remission (whether partial or attending physician and other clinical factors . Dosages for total) , whether detectable or undetectable , or enhancement any one patient depends on many factors , including the or improvement of the condition , disorder or disease . Treat patient's size , body surface area , age , sex , the particular ment includes eliciting a clinically significant response compound to be administered , time and route of adminis without excessive levels of side effects . Treatment also tration , the kind of therapy, general health and other drugs includes prolonging survival as compared to expected sur being administered concurrently . An " effective dose ” refers vival if not receiving treatment. In other embodiments , to amounts of the active ingredient that are sufficient to " treatment” or “ treating” or “ treated ” refers to prophylactic affect the course and the severity of the disease , leading to measures , wherein the object is to delay onset of or reduce the reduction or remission of such pathology and may be severity of an undesired physiological condition , disorder or determined using known methods. In some embodiments , disease, such as, for example is a person who is predisposed the inducible monovalent target -binding proteins described to a disease ( e.g., an individual who carries a genetic marker herein for a disease such as breast cancer ) . [0099 ] In some embodiments of the methods described Methods of Treatment herein , the inducible monovalent target binding protein [0096 ] Also provided herein , in some embodiments , are described herein are administered in combination with an methods and uses for stimulating the immune system of an agent for treatment of the particular disease , disorder or US 2020/0148771 A1 May 14 , 2020 14 condition . Agents include but are not limited to , therapies [0106 ] Mice of 8-14 weeks of age (n = 5-10 ) are subcuta involving antibodies , small molecules ( , chemotherapeu neously inoculated with 2x10 % B16F10 melanoma cells . tics ) , hormones ( steroidal, peptide , and the like ), radiothera Mice which do not receive the full injection , or whose pies (y -rays , X - rays , and /or the directed delivery of radio injection does not result in a visible subcutaneous bleb isotopes , microwaves , UV radiation and the like ) , gene without leakage , are not used in the experiment and sacri therapies ( e.g., antisense , retroviral therapy and the like ) and ficed immediately . other immunotherapies. In some embodiments , the inducible [0107 ] Mice with tumor are injected intraperitoneally with monovalent target binding protein described herein are the inducible monovalent CTLA - 4 binding antibody alone, administered in combination with anti -diarrheal agents , anti or in combination with a second therapeutic agent . Tumor emetic agents , analgesics, opioids and / or non - steroidal anti measurements are taken every other day using digital cali inflammatory agents . In some embodiments , the , the induc pers . ible monovalent target binding protein described herein are [0108 ] Treatment of the mice with the combination of the administered before, during, or after surgery . inducible monovalent CTLA - 4 binding antibody and the [0100 ] According to another embodiment of the invention , second therapeutic agent results in improved attenuation of kits for detecting a cancer , and for diagnosis , prognosis or tumorigenesis with a significant decrease in tumor area , and monitoring are provided . The kits include the foregoing improved survival , compared to a monotherapy with the inducible monovalent target binding protein ( , labeled anti second therapeutic agent alone . immune checkpoint protein inducible monovalent target binding protein or antigen binding fragments thereof) , and Example 3 : Inducible Monovalent CTLA - 4 Binding one or more compounds for detecting the label. In some Antibody Exhibits Reduced Specificity Towards embodiments , the label is selected from the group consisting Cell Line which Overexpresses CTLA - 4 but is of a fluorescent label , an enzyme label , a radioactive label, Protease Deficient a nuclear magnetic resonance active label , a luminescent [0109 ] Cells overexpressing CTLA - 4 and exhibiting low label, and a chromophore label . expression of a matrix metalloprotease are separately incu bated with an exemplary inducible monovalent CTLA -4 EXAMPLES antibody according to the present disclosure , or a control [0101 ] The examples below further illustrate the described non - inducible CTLA - 4 antibody . Cells expressing normal embodiments without limiting the scope of the invention . levels of CTLA - 4 and proteases are also incubated with exemplary inducible monovalent CTLA- 4 antibody accord Example 1 : Treatment with Inducible Monovalent ing to the present disclosure , or a control non - inducible CTLA -4 Binding Antibody Attenuates CTLA - 4 antibody. Tumorigenesis and Enhances Survival [0110 ] Results indicate that in the absence of protease [0102 ] C57B1/ 6 mice are obtained from The Jackson secretion , the inducible antibody of the present disclosure Laboratory. B16F10 murine melanoma cell line is grown in binds the protease expressing cells but does not bind the Iscove's modified Dulbeco's medium supplemented with protease- deficient antigen expressing cells . In contrast, the 10 % FBS , 2 mM L - glutamine , and antibiotics (50 U /ml control non - inducible antibody lacks the ability to selec penicillin and 50 ug /ml streptomycin ). Mice of 8-14 weeks tively bind the protease expressing cells over the protease of age ( n = 5-10 ) are subcutaneously inoculated with 2x105 deficient ones. Thus, the exemplary inducible monovalent B16F10 melanoma cells . Mice which do not eive the full antibody of the present disclosure is advantageous, for injection , or whose injection does not result in a visible example , in terms of reducing off -tumor toxicity . subcutaneous bleb without leakage , are not used in the Example 4 : Characterization of Exemplary experiment and sacrificed immediately . Inducible Monovalent Antibodies of this Disclosure [0103 ] For the treatment of B16F10 melanoma , mice are given intraperitoneal injections (1.p. ) of 50 ug of inducible [0111 ] Expression , Purification and Proteolytic Activation monovalent anti -PD -L1 antibody, or PBS ( vehicle control) of Exemplary Inducible Monovalent Antibodies of this every other day starting day 10 after B16F10 inoculation . Disclosure [ 0104 ] Tumor measurements are taken every other day [ 0112 ] Heavy and light chain sequences for four exem using digital calipers . Results indicate that treatment of plary monovalent antibodies (disclosed as SEQ ID Nos. tumor- bearing mice with the inducible monovalent CTLA - 4 43-50 ) according to this disclosure were cloned into mam binding antibody results in an attenuation of tumorigenesis malian expression vector pcDNA 3.4 ( ThermoFisher ) pre with a significant decrease in tumor area, and the mice show ceded by a leader sequence . Two of the four exemplary prolonged survival. inducible monovalent antibodies were CTLA4 binding inducible monovalent antibodies, derived from parental anti Example 2 : Combination Therapies with an bodies targeting CTLA4 ( ipilimumab , tremelimumab ) , and Inducible Monovalent CTLA - 4 Binding Antibody two were CD40 binding inducible monovalent antibodies and a Second Therapeutic Agent Show Increased derived from two parental antibodies targeting CD40 Effects on Tumorigenesis Attenuation and Survival (dacetuzumab , selicrelumab ). The CTLA4 binding inducible monovalent antibodies are referred to herein as Ipi [0105 ] C57B1/ 6 mice are obtained from The Jackson ProMonoMab and Treme- ProMonoMab , and the CD40 Laboratory. B16F10 murine melanoma cell line is grown in binding inducible monovalent antibodies are referred to Iscove's modified Dulbeco's medium supplemented with herein as Dace -ProMono Mab and Seli - ProMonoMab . 10 % FBS , 2 mM L - glutamine, and antibiotics (50 U /ml Sequences for the four exemplary inducible monovalent penicillin and 50 ug /ml streptomycin ). antibodies are provided in Table I. Each of these antibodies US 2020/0148771 A1 May 14 , 2020 15

contained variable heavy and light domains connected by a Results linker comprising the sequence set forth as SEQ ID No. 51 [0116 ] The Exemplary Inducible Monovalent Antibodies (GSSGGSGGSGGSGLSGRSDNHGSSGT ) . The linker were Specifically Cleaved by a Tumor Associated Protease sequence is in bold font and underlined in Table I. In each In Vitro of these antibody sequences, lysine residues in the Fc region [0117 ] The CTLA4 binding ( Ipi -ProMono Mab and of the heavy chains and aspartic acid residues in the Fc Treme- ProMono Mab ) and CD40 binding ( Dace region of the light chains, are involved in forming heterodi ProMonoMab and Seli - ProMonoMab ) inducible monova meric Fc regions . The lysine residues ( K ) are underlined in lent antibodies were kept in the prodrug form by splitting the bold font, Table I. The aspartic acid residues (D ) are under monovalent functional scFv as two separate non - binding lined in bold font, in Table I. scFvs ( as shown in FIG . 2 ) . The non -binding scFvs were kept inert by keeping the functional VH or VL domains [0113 ] Expi293F cells ( Thermo Fisher ) weremaintained in preferentially paired with non -binding VL and VH domains, suspension between 0.2 to 8E6 cells /ml in Expi293 media . respectively , and tethered via a protease - cleavable linker. Purified plasmid DNA was transfected into Expi293 cells in The anti - respiratory syncytial virus (RSV ) F -protein anti accordance with Expi293 Expression System Kit ( Thermo body , palivizumab , was used as the non -binding VL and VH Fisher ) protocols , and maintained for 4-6 days post trans domains because RSV F - proteins are not normally found in fection . Conditioned media was partially purified by affinity healthy tissues. It was observed that all four inducible and desalting chromatography, polished by ion exchange monovalent antibodies, targeting either CD40 or CTLA4 , chromatography, and concentrated with Amicon Ultra cen were proteolytically activated using the tumor -associated trifugal filtration units (EMD Millipore ) . Fractions pooling protease matriptase (as shown in FIG . 3 ). The SDS- PAGE and final purity were assessed by SDS - PAGE . Purified confirmed that the expected molecular weights of the induc exemplary monovalent antibodies were either proteolyti ible monovalent antibodies in their prodrug forms (, 105.1 cally activated with 40 nM recombinant human matriptase kDa for Dace - ProMonoMa, 105.9 kDa for Treme (R & D Systems) in phosphate -buffered saline (PBS ) or ProMonoMab ) dropped upon proteolytic cleavage when the untreated control with PBS alone for overnight at room antibodies were converted to their active forms ( , in case of temperature . Assessment of proteolytic activation was con Dace- ProMonoMab the molecular weight dropped to about firmed using SDS -PAGE . 77.7 kDa, in case of Treme - ProMono Mab the molecular weight dropped to about 78.6 kda ) . Steady - State Affinity Measurements Proteolytic Activation of the Inducible Monovalent Anti [0114 ] The affinities of the exemplary monovalent anti bodies Increases their Binding Affinity to Target Antigen bodies , in the prodrug and activated forms, were measured [0118 ] Differential binding of the inducible monovalent by an ELISA assay. Briefly , target antigens , CD40 or antibodies in their prodrug and active forms was determined CTLA4 (both from Acro Biosystems) , were coated at 1 by comparing the apparent K , of the untreated (prodrug ) or ug/ ml in a Nunc MaxiSorp plate ( Thermo Fisher ) for 1 hour the protease -treated ( active ) forms of the inducible monova and blocked with SuperBlock ( Thermo Fisher) . The exem lent antibodies to their target antigens in an ELISA assay plary monovalent antibodies, whether intact or matriptase (results shown in FIG . 4 ). Differences in the apparent activated , were added at concentrations ranging from 0.001 affinities of the inducible monovalent antibodies were 5.1x to 1000 nM , as indicated in the x -axis of each plot shown in ( for Dace -ProMono Mab ) , 2.0x ( for Seli -ProMono Mab ) , FIG . 4 , and incubated for 1 hour at room temperature. Plates 6.8 % ( for the Ipi- ProMono Mab ), and 5.3x ( for Treme were washed 3 times using PBS supplemented with 0.05 % ProMonoMab ) before and after matriptase treatment, with Tween - 20 (PBS - T ). Goat anti -human IgG -Fc - peroxidase the matriptase- treated inducible monovalent antibody hav conjugate antibody (Sigma ) was used at 1: 10,000 dilution in ing the higher binding affinity in every instance . PBS - T and incubated for an additional hour. Plates were CD40 Binding Inducible Monovalent Antibodies Demon washed 3 times using PBS - T , followed by development strated Potent CD40 Signaling after Proteolytic Activation [0119 ] In this experiment it was demonstrated that the using TMB substrate ( ThermoFisher ), and neutralizing with inducible monovalent antibodies retain potent activity after 0.1 M sulfuric acid . The resulting colorimetric assay was proteolytic activation in a cell- based functional assay . In measured at 450 nm using SpectraMax spectrophotometer particular, the experiment determined the activity of Seli (Molecular Devices) . ProMonoMab in the untreated (prodrug ) or the matriptase CD40 Signaling Assay treated ( active ) forms, in a CD40 - sensitive reporter cell line ( results shown in FIG . 5 ). The engineered HEK -Blue [0115 ] The agonistic activity of the CD40 binding induc CD40L cell line used in this method secreted alkaline ible monovalent antibodies was determined using a human phosphatase into the media in response to CD40 signaling , CD40 -dependent reporter cell line assay, HEK - Blue CD40L which was subsequently measured using a colorimetric assay ( InvivoGen ) , as per manufacture’s protocol. Briefly , assay . Despite a relatively small 2.0x differential affinity on 293 - Blue cells at 8000 cells per well were mixed with ELISAS, there was a distinct and clear differential activity baseline level of CD40L at 0.1 nM and the CD40 binding between the prodrug and active forms of the Seli inducible monovalent antibodies at the indicated concentra ProMonoMab (FIG . 5 , left panel ) . Because anti -CD40 ago tions , with or without the addition of mouse anti- human Fc nistic antibodies often depend on FcyR -mediated clustering polyclonal antibody at 20 ug/ ml for crosslinking. After of antibodies on cell membranes to correspondingly cluster incubating at 37° C. for 20 hours, the supernatants were CD40 on the opposing cell membrane, an experiment was removed and mixed with QUANTI- Blue (InvivoGen ) at 1: 4 designed to stimulate such clustering effect by adding anti ratio for 3 additional hours at 37 ° C. The resulting colori human IgG polyclonal antibodies to crosslink the Seli metric assay was measured at 640 nm using SpectraMax ProMonoMabs . The addition of crosslinking antibodies to spectrophotometer (Molecular Devices ) . simulate the clustering effect on cell membranes improved US 2020/0148771 A1 May 14 , 2020 16 the sensitivity of the cell assay , and allowed determination 0.37 nM whereas EC50 was not reached for the untreated of a > 405x differential activity . As shown in FIG . 4 , EC50 of Seli -ProMono Mab even at the highest tested concentration the protease -activated form of the Seli -ProMonoMab was of 150 nM . TABLE I Sequences of exemplary inducible monovalent antibodies of this disclosure Dace - ProMonomab DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYOQKPGKAPKLLIYDTSKLASGV VH ( SEQ ID No. 43 ) PSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKGSSGGSGG SGGSGLSGRSDNHGSSGTEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYYTHWVRO APGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYC AREGIYWWGQGTLVTVSSGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTOKSLSLSPG * Dace - ProMonomab QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDD VL ( SEQ ID No. 44 ) KKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGT TVTVSS GSSGGSGGSGGSGLSGRSDNHGSSGTDIQMTQSPSSLSASVGDRVTITCRSSQSLVH SNGNTFLHWYQQKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLOPEDF ATYFCSQTTHVPWTFGQGTKVEIKGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYTLPPSREEMTKNO VSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQ GNVFSCSVMHEALHNHYTOKSLSLSPG * Seli - ProMono Mab VH DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYOQKPGKAPKLLIYDTSKLASGV ( SEQ ID No. 45 ) PSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKGSSGGSGG SGGSGLSGRSDNHGSSGTQVQLVOSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRO APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYC ARDQPLGYCTNGVCSYFDYWGQGTLVTVSSGGGGGDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRK EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPG * Seli - ProMonomab VL QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDD ( SEQ ID No. 46 ) KKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGT TVTVSS GSSGGSGGSGGSGLSGRSDNHGSSGTDIQMTQSPSSVSASVGDRVTITCRASQGIYS WLAWYOQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYC QQANIFPLTFGGGTKVEIKGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFS CSVMHEALHNHYTOKSLSLSPG * Ipi - ProMonoMab VH DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGV ( SEQ ID No. 47 ) PSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKGSSGGSGG SGGSGLSGRSDNHGSSGTQVOLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRO APGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDYWGQGTLVTVSSGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTOKSLSLSPG * Ipi - ProMonoMab VL QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDD ( SEQ ID No. 48 ) KKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGT TVTVSS GSSGGSGGSGGSGLSGRSDNHGSSGTEIVLTQSPGTLSLSPGERATLSCRASQSVGS SYLAWYOQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPWTFGQGTKVEIKGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVF SCSVMHEALHNHYTOKSLSLSPG * Treme - ProMonoMab DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGV VH ( SEQ ID No. 49 ) PSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKGSSGGSGG SGGSGLSGRSDNHGSSGTQVOLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRO APGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDPRGATLYYYYYGMDVWGQGTTVTVSSGGGGGDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTY US 2020/0148771 A1 May 14 , 2020 17

TABLE I - continued Sequences of exemplary inducible monovalent antibodies of this disclosure RVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTOKSLSLSPG * Treme - ProMonoMab QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDD VL ( SEQ ID No. 50 ) KKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGT TVTVSS GSSGGSGGSGGSGLSGRSDNHGSSGTDIQMTQSPSSLSASVGDRVTITCRASQSINS YLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYSTPFTFGPGTKVEIKGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFS CSVMHEALHNHYTOKSLSLSPG *

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 67 < 210 > SEQ ID NO 1 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP7 cleavage domain sequence < 400 > SEQUENCE : 1 Lys Arg Ala Leu Gly Leu Pro Gly 1 5

< 210 > SEQ ID NO 2 < 211 > LENGTH : 40 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP7 cleavage domain sequence < 400 > SEQUENCE : 2 Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu 1 5 10 15 Arg Pro Leu Ala Leu Trp Arg Ser Asp Arg Asp Arg Asp Arg Asp Arg 20 25 30 Asp Arg Asp Arg Asp Arg Asp Arg 35 40

< 210 > SEQ ID NO 3 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP9 cleavage domain sequence < 220 > FEATURE : < 221 > NAME /KEY : MOD_RES < 222 > LOCATION : ( 3 ) .. ( 3 ) < 223 > OTHER INFORMATION : Ser or Thr < 220 > FEATURE : < 221 > NAME / KEY : MOD_RES < 222 > LOCATION : ( 4 ) .. ( 4 ) < 223 > OTHER INFORMATION : Leu or Ile < 220 > FEATURE : < 221 > NAME /KEY : MOD_RES US 2020/0148771 A1 May 14 , 2020 18

- continued < 222 > LOCATION : ( 5 ) .. ( 5 ) < 223 > OTHER INFORMATION : Ser or Thr < 400 > SEQUENCE : 3 Pro Arg Xaa Xaa Xaa 1 5

< 210 > SEQ ID NO 4 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP9 cleavage domain sequence < 400 > SEQUENCE : 4 Leu Glu Ala Thr Ala 1 5

< 210 > SEQ ID NO 5 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP11 cleavage domain sequence < 400 > SEQUENCE : 5 Gly Gly Ala Ala Asn Leu Val Arg Gly Gly 1 5 10

< 210 > SEQ ID NO 6 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP14 cleavage domain sequence < 400 > SEQUENCE : 6 Ser Gly Arg Ile Gly Phe Leu Arg Thr ? 1 5 10

< 210 > SEQ ID NO 7 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 400 > SEQUENCE : 7 Pro Leu Gly Leu Ala Gly 1 5

< 210 > SEQ ID NO 8 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 220 > FEATURE : < 221 > NAME / KEY : MOD_RES < 222 > LOCATION : ( 6 ) .. ( 6 ) < 223 > OTHER INFORMATION : Any amino acid US 2020/0148771 A1 May 14 , 2020 19

- continued

< 400 > SEQUENCE : 8 Pro Leu Gly Leu Ala Xaa 1 5

< 210 > SEQ ID NO 9 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 400 > SEQUENCE : 9 Pro Leu Gly Cys Ala Gly 1 5

< 210 > SEQ ID NO 10 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 400 > SEQUENCE : 10 Glu Ser Pro Ala Tyr Tyr Thr Ala 1 5

< 210 > SEQ ID NO 11 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 400 > SEQUENCE : 11 Arg Leu Gln Leu Lys Leu 1 5

< 210 > SEQ ID NO 12 < 211 > LENGTH : 7 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP cleavage domain sequence < 400 > SEQUENCE : 12 Arg Leu Gin Leu Lys Ala Cys 1 5

< 210 > SEQ ID NO 13 < 211 > LENGTH : 7 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : MMP2 , MMP9 , MMP14 cleavage domain sequence < 220 > FEATURE : < 221 > NAME / KEY : MOD_RES < 222 > LOCATION : ( 3 ) .. ( 3 ) < 223 > OTHER INFORMATION : Citrulline < 220 > FEATURE : < 221 > NAME / KEY : MOD_RES US 2020/0148771 A1 May 14 , 2020 20

- continued < 222 > LOCATION : ( 5 ) .. (5 ) < 223 > OTHER INFORMATION : Homophenylalanine < 400 > SEQUENCE : 13 Glu Pro Xaa Gly Xaa Tyr Leu 1 5

< 210 > SEQ ID NO 14 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Urokinase plasminogen activator (UPA ) cleavage domain sequence < 400 > SEQUENCE : 14 Ser Gly Arg Ser Ala 1 5

< 210 > SEQ ID NO 15 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Urokinase plasminogen activator (UPA ) cleavage domain sequence < 400 > SEQUENCE : 15 Asp Ala Phe Lys 1

< 210 > SEQ ID NO 16 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Urokinase plasminogen activator (UPA ) cleavage domain sequence < 400 > SEQUENCE : 16 Gly Gly Gly Arg Arg 1 5

< 210 > SEQ ID NO 17 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Lysosomal Enzyme cleavage domain sequence < 400 > SEQUENCE : 17 Gly Phe Leu Gly 1

< 210 > SEQ ID NO 18 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Lysosomal Enzyme cleavage domain se ie < 400 > SEQUENCE : 18

Ala Leu Ala Leu US 2020/0148771 A1 May 14 , 2020 21

- continued

1

< 210 > SEQ ID NO 19 < 211 > LENGTH : 2 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Lysosomal Enzyme cleavage domain sequence < 400 > SEQUENCE : 19 Phe Lys 1

< 210 > SEQ ID NO 20 < 211 > LENGTH : 3 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Cathepsin B cleavage domain sequence < 400 > SEQUENCE : 20 Asn Leu Leu 1

< 210 > SEQ ID NO 21 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Cathepsin D cleavage domain sequence < 400 > SEQUENCE : 21 Pro Ile Cys Phe Phe 1 5

< 210 > SEQ ID NO 22 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Cathepsin K cleavage domain sequence < 400 > SEQUENCE : 22 Gly Gly Pro Arg Gly Leu Pro Gly 1 5

< 210 > SEQ ID NO 23 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Prostate Specific Antigen cleavage domain sequence < 400 > SEQUENCE : 23 His Ser Ser Lys Leu Gln 1 5

< 210 > SEQ ID NO 24 < 211 > LENGTH : 7 < 212 > TYPE : PRT US 2020/0148771 A1 May 14 , 2020 22

- continued < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Prostate Specific Antigen cleavage domain sequence < 400 > SEQUENCE : 24 His Ser Ser Lys Leu Gin Leu 1 5

< 210 > SEQ ID NO 25 < 211 > LENGTH : 9 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Prostate Specific Antigen cleavage domain sequence < 400 > SEQUENCE : 25 His Ser Ser Lys Leu Gin Glu Asp Ala 1 5

< 210 > SEQ ID NO 26 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Herpes Simplex virus < 400 > SEQUENCE : 26 Leu Val Leu Ala Ser Ser Ser Phe Gly Tyr 1 5 10

< 210 > SEQ ID NO 27 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Human immunodeficiency virus < 400 > SEQUENCE : 27 Gly Val Ser Gin Asn Tyr Pro Ile Val Gly 1 5 10

< 210 > SEQ ID NO 28 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : CMV Protease cleavage domain sequence < 400 > SEQUENCE : 28 Gly Val Val Gin Ala Ser Cys Arg Leu Ala 1 5 10

< 210 > SEQ ID NO 29 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Thrombin cleavage domain sequence < 220 > FEATURE : < 221 > NAME / KEY : MOD_RES < 222 > LOCATION : ( 2 ) .. ( 2 ) < 223 > OTHER INFORMATION : Piperidine < 400 > SEQUENCE : 29 Phe Xaa Arg Ser US 2020/0148771 A1 May 14 , 2020 23

- continued

1

< 210 > SEQ ID NO 30 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Thrombin cleavage domain sequence < 400 > SEQUENCE : 30 Asp Pro Arg Ser Phe Leu 1 5

< 210 > SEQ ID NO 31 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Thrombin cleavage domain sequence < 400 > SEQUENCE : 31 Pro Pro Arg Ser Phe Leu 1 5

< 210 > SEQ ID NO 32 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Caspase - 3 cleavage domain sequence < 400 > SEQUENCE : 32 Asp Glu Val Asp 1

< 210 > SEQ ID NO 33 < 211 > LENGTH : 5 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Caspase - 3 cleavage domain sequence < 400 > SEQUENCE : 33 Asp Glu Val Asp Pro 1 5

< 210 > SEQ ID NO 34 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Caspase - 3 cleavage domain sequence < 400 > SEQUENCE : 34 Lys Gly Ser Gly Asp Val Glu Gly 1 5

< 210 > SEQ ID NO 35 < 211 > LENGTH : 6 < 212 > TYPE : PRT US 2020/0148771 A1 May 14 , 2020 24

- continued < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Interleukin 1 -beta converting enzyme cleavage domain sequence < 400 > SEQUENCE : 35 Gly Trp Glu His Asp Gly 1 5

< 210 > SEQ ID NO 36 < 211 > LENGTH : 7 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Enterokinase cleavage domain sequence < 400 > SEQUENCE : 36 Glu Asp Asp Asp Asp Lys Ala 1 5

< 210 > SEQ ID NO 37 < 211 > LENGTH : 9 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : FAP cleavage domain sequence < 400 > SEQUENCE : 37 Lys Gin Glu Gln Asn Pro Gly Ser Thr 1 5

< 210 > SEQ ID NO 38 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Kallikrein 2 cleavage domain sequence < 400 > SEQUENCE : 38 Gly Lys Ala Phe Arg Arg 1 5

< 210 > SEQ ID NO 39 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Plasmin cleavage domain sequence < 400 > SEQUENCE : 39 Asp Ala Phe Lys 1

< 210 > SEQ ID NO 40 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Plasmin cleavage domain sequence < 400 > SEQUENCE : 40 US 2020/0148771 A1 May 14 , 2020 25

- continued

Asp Val Leu Lys 1

< 210 > SEQ ID NO 41 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : Plasmin cleavage domain sequence < 400 > SEQUENCE : 41 Asp Ala Phe Lys 1

< 210 > SEQ ID NO 42 < 211 > LENGTH : 7 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : TOP cleavage domain sequence < 400 > SEQUENCE : 42 Ala Leu Leu Leu Ala Leu Leu 1 5

< 210 > SEQ ID NO 43 < 211 > LENGTH : 477 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 43 Asp Ile Gin Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gin Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gin Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Ser Gly Gly Ser 100 105 110 Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly 115 120 125 Ser Ser Gly Thr Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser 145 150 155 160 Phe Thr Gly Tyr Tyr Ile His Trp Val Arg Gin Ala Pro Gly Lys Gly 165 170 175 US 2020/0148771 A1 May 14 , 2020 26

- continued Leu Glu Trp Val Ala Arg Val Ile Pro Asn Ala Gly Gly Thr Ser Tyr 180 185 190 Asn Gin Lys Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Asn Ser Lys 195 200 205 Asn Thr Ala Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Arg Glu Gly Ile Tyr Trp Trp Gly Gin Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly Asp Lys Thr His Thr 245 250 255 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg Glu Glu Gin Tyr Gly Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 370 375 380 Ser Arg Lys Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp 420 425 430 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475

< 210 > SEQ ID NO 44 < 211 > LENGTH : 489 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 44 Gin Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 US 2020/0148771 A1 May 14 , 2020 27

- continued

Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gin Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly 115 120 125 Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser 130 135 140 Gly Thr Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser 145 150 155 160 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gin Ser Leu Val 165 170 175 His Ser Asn Gly Asn Thr Phe Leu His Trp Tyr Gin Gin Lys Pro Gly 180 185 190 Lys Ala Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 195 200 205 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220 Thr Ile Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Phe Cys Ser 225 230 235 240 Gin Thr Thr His Val Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu 245 250 255 Ile Lys Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 260 265 270 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 275 280 285 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 290 295 300 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 305 310 315 320 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 325 330 335 Glu Gin Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 340 345 350 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 355 360 365 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 370 375 380 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 385 390 395 400 Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 405 410 415 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 420 425 430 Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 435 440 445 US 2020/0148771 A1 May 14 , 2020 28

- continued Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 450 455 460 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 465 470 475 480 Gln Lys Ser Leu Ser Leu Ser Pro Gly 485

< 210 > SEQ ID NO 45 < 211 > LENGTH : 489 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 45 Asp Ile Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gin Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gin Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Ser Gly Gly Ser 100 105 110 Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly 115 120 125 Ser Ser Gly Thr Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys 130 135 140 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 145 150 155 160 Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gin Gly 165 170 175 Leu lu Trp Met Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr 180 185 190 Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile 195 200 205 Ser Thr Ala Tyr Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Arg Asp Gin Pro Leu Gly Tyr Cys Thr Asn Gly 225 230 235 240 Val Cys Ser Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val 245 250 255 Ser Ser Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 260 265 270 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 275 280 285 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 290 295 300 US 2020/0148771 A1 May 14 , 2020 29

- continued

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 305 310 315 320 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 325 330 335 Glu Gin Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 340 345 350 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 355 360 365 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 370 375 380 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu 385 390 395 400 Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 405 410 415 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 420 425 430 Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe 435 440 445 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 450 455 460 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 465 470 475 480 Gin Lys Ser Leu Ser Leu Ser Pro Gly 485

< 210 > SEQ ID NO 46 < 211 > LENGTH : 484 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 46 Gin Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gin 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gin Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly 115 120 125 Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser 130 135 140 Gly Thr Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser US 2020/0148771 A1 May 14 , 2020 30

- continued

145 150 155 160 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Tyr 165 170 175 Ser Trp Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Asn Leu 180 185 190 Leu Ile Tyr Thr Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe 195 200 205 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 210 215 220 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ala Asn Ile Phe 225 230 235 240 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly 245 250 255 Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 260 265 270 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 275 280 285 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 290 295 300 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 305 310 315 320 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Gly Ser 325 330 335 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 340 345 350 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 355 360 365 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 370 375 380 Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 385 390 395 400 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 405 410 415 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr 420 425 430 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu 435 440 445 Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 450 455 460 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 465 470 475 480 Leu Ser Pro Gly

< 210 > SEQ ID NO 47 < 211 > LENGTH : 481 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 47 Asp Ile Gln Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Val Gly US 2020/0148771 A1 May 14 , 2020 31

- continued

1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gin Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Ser Gly Gly Ser 100 105 110 Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly 115 120 125 Ser Ser Gly Thr Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val 130 135 140 Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 195 200 205 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220 Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp 225 230 235 240 Gly Gin Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly Asp 245 250 255 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 260 265 270 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 275 280 285 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 290 295 300 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 305 310 315 320 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Gly Ser Thr Tyr Arg 325 330 335 Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys 340 345 350 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 355 360 365 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gin Val Tyr 370 375 380 Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gin Val Ser Leu 385 390 395 400 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 405 410 415 US 2020/0148771 A1 May 14 , 2020 32

- continued

Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 420 425 430 Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 435 440 445 Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 450 455 460 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 465 470 475 480 Gly

< 210 > SEQ ID NO 48 < 211 > LENGTH : 485 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 48 Gin Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gin 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gin Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val T Val Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly 115 120 125 Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser 130 135 140 Gly Thr Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 145 150 155 160 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Gly 165 170 175 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gin Ala Pro Arg 180 185 190 Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg 195 200 205 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 210 215 220 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser 225 230 235 240 Ser Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Gly Gly 245 250 255 Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 260 265 270 US 2020/0148771 A1 May 14 , 2020 33

- continued

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 275 280 285 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 290 295 300 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 305 310 315 320 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Gly 325 330 335 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 340 345 350 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 355 360 365 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu 370 375 380 Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 385 390 395 400 Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 405 410 415 Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Asp Thr 420 425 430 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp 435 440 445 Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 450 455 460 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 465 470 475 480 Ser Leu Ser Pro Gly 485

< 210 > SEQ ID NO 49 < 211 > LENGTH : 488 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gin Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Ser Gly Gly Ser 100 105 110 Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly US 2020/0148771 A1 May 14 , 2020 34

- continued

115 120 125 Ser Ser Gly Thr Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val 130 135 140 Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 195 200 205 Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr 225 230 235 240 Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro 260 265 270 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 275 280 285 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 290 295 300 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 305 310 315 320 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 325 330 335 Gin Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 340 345 350 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 355 360 365 Ala Leu Pro Ala Pro Il Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 370 375 380 Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met 385 390 395 400 Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 405 410 415 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 420 425 430 Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu 435 440 445 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 450 455 460 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 465 470 475 480 Lys Ser Leu Ser Leu Ser Pro Gly 485

< 210 > SEQ ID NO 50 < 211 > LENGTH : 484 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence US 2020/0148771 A1 May 14 , 2020 35

- continued

< 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 400 > SEQUENCE : 50 Gin Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gin 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gin Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly 115 120 125 Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser 130 135 140 Gly Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 145 150 155 160 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Asn 165 170 175 Ser Tyr Leu Asp Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu 180 185 190 Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe 195 200 205 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 210 215 220 Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Tyr Ser Thr 225 230 235 240 Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Gly Gly Gly 245 250 255 Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 260 265 270 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 275 280 285 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 290 295 300 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 305 310 315 320 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Gly Ser 325 330 335 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 340 345 350 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 355 360 365 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro US 2020/0148771 A1 May 14 , 2020 36

- continued 370 375 380 Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin 385 390 395 400 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 405 410 415 Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Asp Thr Thr 420 425 430 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu 435 440 445 Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 450 455 460 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 465 470 475 480 Leu Ser Pro Gly

< 210 > SEQ ID NO 51 < 211 > LENGTH : 26 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 51 Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly 1 5 10 15 Arg Ser Asp Asn His Gly Ser Ser Gly Thr 20 25

< 210 > SEQ ID NO 52 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : cleavage domain sequence < 400 > SEQUENCE : 52 Gly Pro Leu Gly Ile Ala Gly Gin 1 5

< 210 > SEQ ID NO 53 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : cleavage domain sequence < 400 > SEQUENCE : 53 Ala Val Arg Trp Leu Leu Thr Ala 1 5

< 210 > SEQ ID NO 54 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Unknown < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Unknown : cleavage domain sequence US 2020/0148771 A1 May 14 , 2020 37

- continued < 400 > SEQUENCE : 54 Arg Arg Arg Arg Arg Arg 1 5

< 210 > SEQ ID NO 55 < 211 > LENGTH : 20 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 220 > FEATURE : < 221 > NAME /KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. ( 20 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 "Gly Ser " repeating units < 400 > SEQUENCE : 55 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 Gly Ser Gly Ser 20

< 210 > SEQ ID NO 56 < 211 > LENGTH : 30 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 220 > FEATURE : < 221 > NAME / KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. ( 30 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 "Gly Gly Ser " repeating units < 400 > SEQUENCE : 56 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30

< 210 > SEQ ID NO 57 < 211 > LENGTH : 40 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 220 > FEATURE : < 221 > NAME / KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. (40 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 "Gly Gly Gly Ser" repeating units < 400 > SEQUENCE : 57 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly Ser 35 40

< 210 > SEQ ID NO 58 US 2020/0148771 A1 May 14 , 2020 38

- continued < 211 > LENGTH : 40 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 220 > FEATURE : < 221 > NAME / KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. ( 40 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 "Gly Gly Ser Gly " repeating units < 400 > SEQUENCE : 58 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 20 25 30 Gly Gly Ser Gly Gly Gly Ser Gly 35 40

< 210 > SEQ ID NO 59 < 211 > LENGTH : 50 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 220 > FEATURE : < 221 > NAME / KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. (50 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 "Gly Gly Ser Gly Gly " repeating units < 400 > SEQUENCE : 59 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly 50

< 210 > SEQ ID NO 60 < 211 > LENGTH : 50 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic polypeptide < 220 > FEATURE : < 221 > NAME / KEY : MISC_FEATURE < 222 > LOCATION : ( 1 ) .. (50 ) < 223 > OTHER INFORMATION : This sequence may encompass 1-10 " Gly Gly Gly Gly Ser " repeating units < 400 > SEQUENCE : 60 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 US 2020/0148771 A1 May 14 , 2020 39

- continued Gly Ser 50

< 210 > SEQ ID NO 61 < 211 > LENGTH : 20 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 61 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20

< 210 > SEQ ID NO 62 < 211 > LENGTH : 15 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 62 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15

< 210 > SEQ ID NO 63 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 63 Pro Leu Gly Leu Trp Ala 1 5

< 210 > SEQ ID NO 64 < 211 > LENGTH : 6 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence: Synthetic peptide < 400 > SEQUENCE : 64 Arg Val Leu Ala Glu Ala 1 5

< 210 > SEQ ID NO 65 < 211 > LENGTH : 10 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 65 Glu Asp Val Val Cys Cys Ser Met Ser Tyr 1 5 10 US 2020/0148771 A1 May 14 , 2020 40

- continued

< 210 > SEQ ID NO 66 < 211 > LENGTH : 8 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 66 Gly Gly Ile Glu Gly Arg Gly Ser 1 5

< 210 > SEQ ID NO 67 < 211 > LENGTH : 4 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic peptide < 400 > SEQUENCE : 67 Gly Phe Leu Gly 1

1. An inducible monovalent target- binding protein , 7. The inducible monovalent target- binding protein of wherein the protein comprises a first polypeptide chain and claim 1 , wherein the Fcl and the Fc2 each independently a second polypeptide chain , comprise one or more amino acid substitutions favoring the first polypeptide chain comprising : a VH target formation of a heterodimeric Fc region . binding domain (VH ), an inactive VL domain ( iVL ) 8.- 9 . (canceled ) that binds to the VH domain , a linker (L1 ) , and a first 10. The inducible monovalent target- binding protein of monomeric Fc domain (Fc1 ) comprising a CH3 and a claim 1 , wherein the iVH and the iVL each independently CH2 domain ; comprises one or more mutations that prohibit targetbinding the second polypeptide chain comprising : a VL target of said domains . binding domain ( VL ) , an inactive VH domain (iVH ) 11. The inducible monovalent target -binding protein of that binds to the VL domain , a linker (L6 ) , a second claim 1 , wherein the VH , the iVL , and the L1 of the first monomeric Fc domain (Fc2 ) comprising a CH3 and a polypeptide form a scFv . CH2 domain ; 12. The inducible monovalent target- binding protein of wherein the iVL and the iVH each comprise at least one claim 1 , wherein the VL , the iVH , and the L6 of the second protease cleavage site , wherein upon activation by protease polypeptide form a scFv. cleavage of the at least one protease cleavage site of both the 13.- 22 . (canceled ) iVL and the iVH , the VH and the VL associate to form an 23. The inducible monovalent target- binding protein of active target -binding domain , and wherein the CH3 domains claim 1 , wherein the active target - binding domain binds to of the Fcl and Fc2 form a heterodimer . a target expressed on a tumor cell . 2. (canceled ) 24. The inducible monovalent target- binding protein of claim 1, 9 wherein the active target -binding domain binds to 3. The inducible monovalent target- binding protein of a tumor antigen . claim 1, wherein the iVL comprises a CDRL1, CDRL2 , and 25. The inducible monovalent target -binding protein of CDRL3 , and the at least one protease cleavage site of the claim 1 , wherein the active target- binding domain binds to iVL is located within the CDRL1, CDRL2 , or CDRL3 . an immune checkpoint protein . 4. The inducible monovalent target- binding protein of 26. The inducible monovalent target- binding protein of claim 1 , wherein the iVH comprises a CDRH1 , CDRH2, and claim 1 , wherein the active target - binding domain binds to CDRH3, and the at least one protease cleavage site of the CD27 , CD40 , OX40 , GITR , CD137 , B7 , CD28 , ICOS , iVH is located within the CDRH1, CDRH2, or CDRH3. A2AR , B7 -H3 , B7 -H4 , BTLA , CTLA - 4 , IDO , KIR , LAG3, 5. The inducible monovalent target- binding protein of PD - 1 , PD -L1 , TIM - 3 , VISTA . claim 1 , wherein the first polypeptide chain comprises a 27. The inducible monovalent target -binding protein of further protease cleavage site , and wherein the second claim 1 , wherein the at least one protease cleavage site of the polypeptide comprises a further protease cleavage site . first polypeptide and the at least one protease cleavage site 6. The inducible monovalent target- binding protein of is independently recognized by at least one of a serine claim 5 , wherein the further protease cleavage site of the first protease, a cysteine protease, an aspartate protease , a threo polypeptide chain is located within Li, and the further nine protease , a glutamic acid protease, a metalloproteinase , protease cleavage site of the second polypeptide chain is a gelatinase, and a asparagine peptide lyase . located within L6 . 28. (canceled ) US 2020/0148771 A1 May 14 , 2020 41

29. An inducible monovalent target- binding protein , 30.- 35 . (canceled ) wherein the protein comprises a first polypeptide chain and 36. The inducible monovalent target- binding protein of a second polypeptide chain , claim 29 , wherein the iVH and the iVL each independently the first polypeptide chain comprising : a VH target comprise one or more mutations that prohibit target binding binding domain (VH ) , an inactive VL domain ( VL ) of said domains . that binds to the VH domain , a linker (L1 ) , and a first 37. The inducible monovalent target -binding protein of monomeric Fc domain ( Fc1) comprising a CH3 and a claim 29 , wherein the VH , the iVL , and the L1 of the first CH2 domain ; polypeptide form a scFv . the second polypeptide chain comprising : a VL target 38. The inducible monovalent target -binding protein of binding domain (VL ), an inactive VH domain (iVH ) claim 29 , wherein the VL , the iVH , and the L6 of the second that binds to the VL domain , a linker (L6 ) , a second polypeptide form a scFv . monomeric Fc domain (Fc2 ) comprising a CH3 and a 39.- 53 . ( canceled ) CH2 domain ; 54. The inducible monovalent target -binding protein of wherein the L1 and L6 each comprise at least one protease claim 1 , wherein the VH comprises a sequence selected from cleavage site , wherein upon activation by protease cleavage the group consisting of SEQ ID Nos . 43 , 45 , 47 , and 49. of the at least one protease cleavage site of both L1 and L6 , 55. The inducible monovalent target - binding protein of the VH and the VL associate to form an active target - binding claim 1 , wherein the VL comprises a sequence selected from domain , and wherein the CH3 domains of the Fcl and Fc2 the group consisting of SEQ ID Nos. 44 , 46 , 48 , and 50 . form a heterodimer.