The Importance of Immunopharmacology in Non- Clinical Safety Assessment of Immune-Oncology Biotherapeutics

The importance of immunopharmacology in non- clinical safety assessment of immune-oncology biotherapeutics

Greg Bannish, Ph.D. SOT-PBSS Joint Spring Symposium: Immuno-oncology: Opportunities, Therapeutic Approaches, and Safety Considerations Friday, April 27th, 2018

+ Immunostimulatory biotherapeutics benefit from immune assessment during preclinical safety evaluation. + An overview of immune analysis are detailed, including development of a sensitive T cell dependent antibody response (TDAR) model which can detect immunostimulatory drug effects. + An example of some unpublished BCMA expression on B cells in cynomolgus monkeys is presented to stress the importance understanding the species immune system and/or drug pharmacology, during preclinical safety assessment. + Best practices for safety assessment of immunostimulatory biotherapeutics

2 Outline

1 Introduction

2 Immunostimulatory TDAR

3 BCMA expression on Cynomolgus B cells

4 Immune oncology safety

3 1. Introduction

4 Introduction

+ Biotherapeutics + Immune system and tolerance

5 Biotherapeutics

6 What are biologics?

+ Term “biologics” covers a wide variety of product classes + mAbs (including domain Abs / fragments / ADCs) + Recombinant proteins and replacement products + Oligonucleotides + Vaccines + Advanced therapies + ATMPs (advanced therapy medicinal products) + Cellular and gene therapy products (CGTs) + Complex + Typically made from cells, cell products, or cells/viruses themselves. + Large + Immunogenicity + Opposite = NCE (new chemical entity) + Small, chemically synthesized

7 Biologics are big!

New chemical Biologic Biologic entity (NCE) (mAb) (Advanced therapy)

aspirin antibody B Cell

Small molecule Large molecule Enormous!!

180 Da ~10 nM 7 uM 150kDa

8 Biologics are big!

Novel chemical Biologic (mAb) Biologic entity (NCE) (Advanced therapy)

Central Park, NYC

NEJM 2011 Reth, Nat Imm 14(8):765-7, 2013 9 Immune system and tolerance

10 Function of the immune system

+ The main role of the immune system is to protect the body from infection + Variety of cell types and systems in place to effectively identify and remove pathogens before they can cause damage to the immune system + Two main arms of the immune system + Innate immune system + Adaptive immune system + Humoral: B cell antibody responses + Cellular: T cell cytotoxic killing + Distinguishes “self” from “non-self”

11 Immune tolerance: self vs non-self

+ Mediated via tolerance mechanisms + Central tolerance + Selection of T and B cells meeting specific criteria, mediated by specific antigen presenting cell populations in the thymus or bone marrow. + Leads to deletion of cells that are under or over-active + Ensures that only cells with appropriate responses to self and non-self antigens will be released into the periphery + Recognition of non-self antigens to mediate adequate surveillance + Limited responses to self antigens to prevent damage of organs and tissues + Peripheral tolerance + Additional control mechanisms exist in lymphoid organs to further refine responses to self and non-self antigens + Limits collateral damage and ensures that immune system reverts to baseline once the pathogen is removed + Exogenous control mechanisms: Regulatory T cells, inhibitory cytokines + Endogenous control mechanisms: induction of anergy or exhaustion

12 Immune system balance

+ For effective immune function, it is critical that there is a BALANCE between identifying pathogens and controlling subsequent responses

Identification and removal of pathogens Identification of tumour cells Controlled and self-limiting responses

Immunosuppression Excessive stimulation Increased risk of infection Hypersensitivity Decreased tumour Autoimmunity surveillance

Normal

13 2. Immunostimulatory TDAR

14 Introduction to the TDAR assay

+ T cell dependent antibody response (TDAR) + Immunize an animal with an antigen, measure the humoral (antibody) immune response + Requires functional immune system: T cells, B cells, and antigen presenting cells (APCs) + Importance + Evaluates the functionality of the adaptive humoral immune system + The most important functional assay for immunotoxicology + Issues + Does not evaluate effect upon the cell-mediated or innate immune system + Not standardized, either immunization or analytical evaluation + Difficult to interpret: high variability, IgM vs IgG, 1o vs 2o responses, etc. + May not detect weaker biological immunosuppressants + Can’t determine immunostimulation (response may be maximal) Introduction to the TDAR assay *

IgM IgG + Antigen: KLH + 3-month study 100 IgG + Immunize: 1° and 2° IgM 80 + Primary: 60 + IgM: max response 7-14 days + IgG: max response 21-28 40 days 20 + Secondary: - + IgM: minimal response

% max % response - 20 40 60 80 + IgG: max response 5-10 days post secondary challenge. KLH KLH

Days The rodent TDAR assay

+ 1979 - A "tiered" approach for detecting immunotoxic agents + Dean et al, Assessment of immunobiological effects induced by chemicals, drugs or food additives. I. Tier testing and screening approach. Drug Chem Toxicol, 1979. 2(1-2): p. 5- 17. + 1988: NTP: TDAR assay with SRBCs to be one of the best predictors of immunotoxicity + Luster et al, Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program's guidelines for immunotoxicity evaluation in mice. Fundam Appl Toxicol, 1988. 10(1): p. 2-19 + 1992: Tested 51 chemicals to determine that IgM splenic B cells AFC/PFC assay (TDAR with SRBCs) is most sensitive predictor of immunotoxicity + Luster et al, Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam Appl Toxicol, 1992. 18(2): p. 200-10 + 1996: EPA - Pesticide testing to require TDAR with SRBCs + EPA, OPPTS 880.3550 Immunotoxicity. Biochemicals test guidelines, EPA, Editor 1996, The Federal Bulletin Board. p. 1-12. 18 Introduction to the TDAR assay

+ Increase in biological therapeutics + Require testing in a pharmacologically relevant species + Many target the immune system + Guidelines + ICH, ICH Guideline S8: Immunotoxicity Studies for Human Pharmaceuticals, U.S.D.o.H.a.H. Services, et al., Editors. 2006: Rockville, MD. p. 1-16. + ICH S6(R1): Preclinical safety evaluations of biotechnology-derived ICH, EMA/CHMP/ICH/731268/1998, ICH guideline S6 (R1) – preclinical safety evaluation of biotechnology-derived pharmaceuticals, U.S.D.o.H.a.H. Services and F.a.D.A. (FDA), Editors. 2011. p. 1-22. The non-human primate TDAR assay

+ 1976: Immunoglobulins in NHP sera + Cole, M.F. and W.H. Bowen, Immunoglobulins A, G, and M in serum and in some secretions of monkeys (Macaca fascicularis syn. irus). Infect Immun, 1976. 13(5): p. 1354-9. + 2005: Assay development of TDAR responses + Piccotti et al, T-cell-dependent antibody response: assay development in cynomolgus monkeys. J Immunotoxicol, 2005. 2(4): p. 191-6. + 2007: Immunotoxicity testing in non-rodent species + Haggerty, H.G., Immunotoxicity testing in non-rodent species. J Immunotoxicol, 2007. 4(2): p. 165-9. + 2011: Inter-laboratory analysis of TDAR in NHPs + Lebrec et al, An inter-laboratory retrospective analysis of immunotoxicological endpoints in non-human primates: T-cell-dependent antibody responses. J Immunotoxicol, 2011. 8(3): p. 238-50. Considerations for TDAR

+ Time points? + Antigen choice? + Adjuvant inclusion? + Secondary immunizations, if and when? + Group males and female data together or report separately? + Immunosuppressant controls? + Which assay? + Interpretation? Include a 2° challenge?

+ More intense, mostly IgG IgM IgG + Requires affinity maturation,

100 isotype switching, memory B IgG cell formation, Th-B cell IgM 80 interactions + Requires at least a 6 week 60

study 40

+ Inability to generate a robust 20 2° IgG response would be a cause for concern -

% max % response - 20 40 60 80

KLH KLH

Days Time points for serum collection

Antigen/Test Study IgM days IgG days + The more, the better! article Day post KLH post KLH + IgM max response: -5 -7 -7 + 7-14 days TA 1 1 1 Antigen 2 + IgG max response: (KLH) + 21-25 days 7 5 + At least 1 pretest (2 better) 9 7 7 12 10 + For 2° immunization 16 14 14 + max:4-7 days 19 17 17 + wait 4-6 weeks or more from 1° 23 21 immunization 25 23 29 27 27 Immunosuppressant controls: worth the effort?

+ An animal model should have appropriate controls to verify performance + TDAR responses: require T, B and APC functions + NCE immunosuppressants work, biologicals better + Strong immunosuppressants ok (FK506, rituximab, etc.), targeted biological immunosuppressants better + A strong immunosuppressant is less able to evaluate/optimize TDAR immunization strategies Immunosuppressants

+ FK506 (NCE) + Acts on T cells + 0.75 mg/kg/day, oral gavage (sub-maximal) + Methotrexate (NCE) + Folate analog inhibitor of DHFR, blocks purine metabolism + Inhibits lymphocyte proliferation, causes apoptosis of active T cells + 1 mg/kg Subcut., once per week + Abatacept (CTLA4-Ig) (biologic) + Blocks co-stimulatory pathway interaction of CD28/CTLA4 on T cells with CD80/86 on APCs + 8 mg/kg, once per week Immunosuppressants

+ Abatacept (CTLA4-Ig) modulates the immune Dendritic cell response by binding to

CD80/86 on APCs, thereby CD80 preventing costimulatory binding of CD28 on naïve T cells and attenuating T cell activation + Administration at 8 mg/kg

2x/wk sufficient to prevent T cell a primary immune response in cynomolgus + Secondary response? Prior TDAR studies at Envigo

Standard TDAR DTH Antigen KLH (10 mg) KLH (1 mg) TT (6 LFU) C. Alb. (625 PNU) ROA SubCut. SubCut. Adjuvant No Yes, IFA # injections 1 3-5 over 2 weeks Duration 1 month >3 months Immuno-suppressant FK506 Abatacept (0.75 mg/kg/day) (8 mg/kg) ?? Oral gavage I.V. daily Once/week Result Reduction, not elimination No decrease DTH study: anti-KLH *

Units/mL II

• 1° response : max 15 days post immunization, in Group Dose Group I only • 2° response: Group I peaks at Day 56, Group II 1 vehicle continues to rise 2 CTLA4-Ig DTH study: anti-TT recall response *

II Units/mL

• Animals respond to TT challenge on Days 1-7 • No increase in titers following challenge on Day 41 • Abatacept does not reduce response New TDAR study

+ Time: 3 months to allow time for 1° response to lessen, and to challenge with a 2° immunization + Less KLH: 100 ug I.D. vs 10 mg Subcut. + Includes an NCE and biologic immunosuppressant + Submaximal responses expected  detection of suppression or stimulation + More time points  bracket response + T-dep: KLH (naïve) and TT (recall) antigens + T-indep.: DNP-LPS (Ti-1) and TNP-ficoll (Ti-2) + Analytical evaluation: titer based ELISAs Additional evaluations

+ T cell mediated immunity: IFN-g ELISpot + Plasma B lymphocyte frequency: Anti-KLH ELISpot + Immunophenotyping: Flow cytometry + Standard panel: tot T, CD4T, CD8T, tot B, NK, mono + Additional: activation, intracellular, other subsets, etc. + Hematology/clinical chemistry parameters Current TDAR *

DNP- TNP- DNP- TNP- Antigen KLH TT LPS Ficoll LPS KLH TT Ficoll All

Flow Sera

Month 1 2 3

•3 month cyno study Group Dose •Immunosuppressed groups 1 vehicle •Immunize with 4 antigens, 1 and 2 each 2 MTX •KLH, TT, DNP-LPS and TNP-Ficoll 3 Abatacept Anti-KLH, IgM

Assay Group

Average of Titer Assay Animal 100,000

Average of Titer 80,000 Animal 60,000 5003M 70,000 5004M 40,000 I 5006M 60,000 20,000 5009M 0 50,000 1 15 22 29 36 50 60 67 74 81 91 Day Group

40,000 Assay Group 1 Average of Titer 100,000

30,000 2 80,000 Animal 20,000 60,000 5015M 3 II 5016M 40,000 5021M

10,000 20,000 5022M Inverse titers Inverse 0 0 1 15 22 29 36 50 60 67 74 81 91 Day 1 15 22 29 36 50 60 67 74 81 91 Assay Group

Average of Titer Day 100,000

80,000 Animal III 60,000 5024M 5026M • 1° response : max 15 days post immunization 40,000 5027M • 2° response: less intense 20,000 9955M 0 1 15 22 29 36 50 60 67 74 81 91 • MTX: reduced 1° response Day • Abatacept: reduced 1° and 2° responses Anti-KLH, IgG: 1° and 2° responses *

Assay Group

Assay Animal Average of Titer 350,000 Average of Titer 300,000 350,000 250,000 Animal 200,000 5003M 5004M 150,000 300,000 5006M I 100,000 5009M 50,000 250,000 0 Group 1 15 22 29 36 50 60 67 74 81 91

Day 200,000 1 Assay Group

Average of Titer 150,000 2 350,000 300,000 100,000 3 250,000 Animal 200,000 5015M II 5016M 150,000 5021M 50,000 100,000 5022M

Inverse titers Inverse 50,000 0 0 1 15 22 29 36 50 60 67 74 81 91 1 15 22 29 36 50 60 67 74 81 91 Day

Assay Group

Day Average of Titer 350,000 300,000

250,000 Animal III 200,000 5024M 5026M 150,000 5027M • 1° response : max 15 days post immunization 100,000 9955M 50,000 • 2° response: more intense 0 1 15 22 29 36 50 60 67 74 81 91 • MTX: reduced 1° and 2° response Day • Abatacept: very low 1° and 2° responses Anti-TT, IgG: recall responses

Assay Group

Average of Titer 70,000 Assay Animal 60,000 50,000 Average of Titer 40,000 Animal I 5003M 30,000 70,000 20,000 5004M 10,000 60,000 0 1 15 22 29 36 50 60 67 74 81 91 50,000 Day Group Assay Group 40,000 Average of Titer 1 70,000 30,000 60,000 2 50,000 40,000 Animal 20,000 II 5015M 3 30,000 20,000 5016M

10,000 10,000 Inverse titers Inverse 0 0 1 15 22 29 36 50 60 67 74 81 91 Day 1 15 22 29 36 50 60 67 74 81 91 Assay Group

Average of Titer Day 70,000 60,000 50,000 40,000 Animal III 30,000 5024M 20,000 5026M 10,000 0 1 15 22 29 36 50 60 67 74 81 91 • Animals respond to TT challenge on Day 15 Day • No increase in titers following challenge on Day 71 • Abatacept and MTX do not reduce response Immunostimulatory drug leads to increased IgG TDAR

Deleted…

35 3. BCMA expression in cynomolgus monkey tissues

36 BCMA expression in cynomolgus monkeys: why?

+ BCMA (CD269) is a tumour marker for multiple myeloma + Multiple Myeloma (MM) is the second most common hematologic malignancy, caused by proliferation of monoclonal plasma cells. + Many companies targeting BCMA with drugs + i.e.: anti-BCMA ADC, CART targeting BCMA, BCMA-T cell bispecific antibody EM801, bispecific T-cell (Bite) targeting BCMA. + BCMA:important biology to understand B cells / humoral immune system + BCMA in cynomolgus monkeys: + Is it restricted to plasma B cells? + Surface expression or intracellular? + Tissue expression (blood, spleen, bone marrow, etc.)? + Similar to humans? Mice? + Do human antibody reagents bind cynomolgus BCMA?

37 BCMA

+ B cell maturation antigen (BCMA). + BCMA: + TNF receptor superfamily member + Other related receptors: TACI and BAFFR + Ligands BAFF and April + Ligand binding stimulates survival and secretion of Ig + Expression on plasma B cells (cell surface and intracellular (Golgi)). Soluble BCMA upregulated in MM patients. + Plasma B cells: + Rare: ~1% of B cells in whole blood. + Expressed in bone marrow and spleen

38 BCMA homology

39 Staining panel

+ CD20, CD40: pan B + Late stage B cell markers: CD38, CD138, CD27 + Procedure: + RBC-depleted whole blood, blocked with mouse serum, stained. At least 1x10^6 CD45+ cells were acquired on a BD Canto II cytometer.

FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 Fluorochrome FITC PE PerCP-Cy5.5 PE-Cy7 APC APC-Cy7 V450 BV510 Panel 1 CD38 CD2/CD159a CD27 CD269 (PE-Vio770) CD138 (MI15) CD20 CD45 CD40 Panel 2 CD38 CD2/CD159a CD27 CD269 (PE-Vio770) CD138 (DL101) CD20 CD45 CD40 Panel 3 CD38 CD2/CD159a CD27 CD138 (MI15) CD269 (REA315) CD20 CD45 CD40 Panel 4 CD38 CD2/CD159a CD27 CD138 (DL101) CD269 (REA315) CD20 CD45 CD40 Panel 5 CD38 CD2/CD159a CD27 CD138 (MI15) CD269 (BAF 193) CD20 CD45 CD40 Panel 6 CD38 CD2/CD159a CD27 CD138 (DL101) CD269 (BAF 193) CD20 CD45 CD40

40 Gating strategy (cynomolgus peripheral blood)

+ General gating strategy for identify B cell and PC. B cells are CD20+/CD2- /CD159a- whereas the PC are further characterized from the CD20-/CD2-/CD159a- population

CD20+ B

CD20- B (Plasma B)

41 Gating strategy: Mauritian cynomolgus

CD27- CD27+

PBMCs

Bone marrow

42 Cynomolgus expression of BCMA in whole blood (clone BAF193)

43 BCMA: preliminary results

+ CD138 (Clone DL-101) can work well under certain conditions: PBMC and bone marrow, but was not robust in whole blood + Unlike human, Vietnamese, Chinese and Mauritian Cynomolgus monkeys’ plasma cells predominantly appear in the CD27- population + CD269 antibody clone BAF 193 appears to specifically stain for CD269 in cynomolgus monkeys + More consistent when cells were intracellular stained for CD269 expression. Higher intracellular expression of BCMA in cyno vs human?

44 4. Preclinical safety of immune- stimulatory IO biologics

45 Biologics (mAB) vs NCE toxicity

+ Fundamentally different approach + NCE: off-target toxicities, genetox, DMPK, safety pharm, no immunogenicity + Biologics: on-target toxicities, no genetox, no DMPK(?), little safety pharm, potential for immunogenicity + ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals + Exaggerated pharmacology source of toxicity + Immunopharmacology + ADA: reduce/neutralize drug exposure + May try dosing through ADA to maintain/preserve exposure + Cytokine release / TeGenero + Hypersensitivity: not common + “Off target” toxicity very rare: + Platelet activation; Everds etal Tox Path 40(6):899-917(2012)

46 Biologics (mAB) vs NCE toxicity: Results

+ Little toxicity found in mAb biologics + dosed at very high levels (ie >100mg/kg, >100x max pharm. dose) + Known toxicities considered: + Cytokine release + Cross-reactive immunogenicity

47 Biologics toxicity: importance of immune system

+ Immune system can affect all biologics via ADA, causing lack of exposure through clearance and/or neutralization. + Immune system can cause toxicity: + Autoimmunity + Hypersensitivity + Cytokine release + Immune complexes + Complement activation + Do immunostimulatory IO biologics have greater risk for toxicity? A narrower therapeutic window? + Is there immune system etiology for the adverse events? + Do the adverse immunological events predict clinical outcomes?

48 Immunostimulatory IO biologics  toxicity

+ Immunostimulatory IO biologics + Targeting multiple checkpoints + Bispecific antibodies targeting T cells and tumour antigens + More potent stimulation of adaptive immune response + Increased frequency of adverse events, likely immune- mediated. + Different approaches from Sponsors + Exploratory toxicology studies to evaluate PD effects in species. + Immunological endpoints on pivotal GLP toxicology studies + Pharmacodynamic endpoints on toxicological studies + Dose levels: how much is needed? + 10x clinical exposure + Attempt to observe adverse event (NOAEL) + ICHS9: 1/6th the highest non-severely toxic dose (HNSTD) in non-rodent + US vs Europe vs Far East?

49 Biologics toxicity: importance of immune system

+ Do immunostimulatory IO biologics have greater risk for toxicity? A narrower therapeutic window? + Yervoy: toxicity on cyno studies correlated but under-represented human adverse events + Is there immune system etiology for the adverse events? + Do the adverse immunological events predict clinical outcomes?

50 Best practices for IO immunostimulatory biologics?

+ Probably not a single correct approach, but several ways to improve chances for success: + Understand your pharmacology + In vitro analysis, pilot animal studies + Receptor occupancy + Monitor pharmacodynamic effects + Assess immune status + Cytokines, complement, immune cell subsets + Anti-drug antibody + Assess immune function + TDAR, ELISpot IFN-g, tetramer staining, etc. + Be ready: contingency plans in protocol for immune evaluation of animals with unscheduled deaths on study.

51 4. Summary

52 Summary

+ Immune oncology drugs are providing a new approach to treating cancer + More efficacious  more dangerous + Preclinical safety can predict clinical adverse events + Understanding of drug pharmacology and immunology within the animal species is important for optimizing quality of safety assessment. + Newer models, analytical tools, and biology knowledge to better assess immune function in toxicology studies.

53 Thank you!

envigo.com/abs2018