Autologous Lymphocyte Infusion Supports Tumor Antigen Vaccine-Induced Immunity in Autologous Stem Cell Transplant for Multiple Myeloma Adam D

Author Manuscript Published OnlineFirst on February 11, 2019; DOI: 10.1158/2326-6066.CIR-18-0198 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Autologous lymphocyte infusion supports tumor antigen vaccine-induced immunity in autologous stem cell transplant for multiple myeloma Adam D. Cohen1†, Nikoletta Lendvai2,3†, Sarah Nataraj4, Naoko Imai4, Achim A. Jungbluth2, Ioanna Tsakos2, Adeeb Rahman4, Anna Huo-Chang Mei4, Herman Singh4, Katarzyna Zarychta4, Seunghee Kim-Schulze4, Andrew Park5, Ralph Venhaus5, Katherine Alpaugh6, Sacha Gnjatic4‡, and Hearn J. Cho4‡

1Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 2Memorial Sloan-Kettering Cancer Center, New York, NY 3Department of Medicine, Weill Medical College of Cornell University, New York, NY 4Tisch Cancer Institute, Icahn School of Medicine at Mt. Sinai, New York, NY 5Ludwig Institute for Cancer Research, New York, NY 6Fox Chase Cancer Center, Philadelphia, PA †These authors contributed equally to this report ‡These authors contributed equally to this report Running Title: Enhancing autologous SCT for multiple myeloma Key Words: Autologous stem cell transplantation, multiple myeloma, cancer immunotherapy, MAGE-A3 Financial Support: The study was sponsored by the Ludwig Institute for Cancer Research (NY, NY), with funding support from GlaxoSmithKline Biologicals SA. S.G. acknowledges funding support for immunological monitoring from the Cancer Research Institute. Conflict of Interest Disclosure Statement: The authors declare no competing financial interests Corresponding Author: Adam D. Cohen. Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., PCAM-2W, Philadelphia PA 19104. Phone: 215-615-5853. Fax: 215- 615-5887. Email: [email protected] Abstract: 247 words Text: 4412 words Figures: 5 Tables: 1 Supplementary Figures: 5 Supplementary Tables: 3

Downloaded from cancerimmunolres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 11, 2019; DOI: 10.1158/2326-6066.CIR-18-0198 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Autologous stem cell transplant (autoSCT), the standard consolidation therapy for multiple

myeloma, improves disease-free survival but is not curative. This could be an ideal setting for

immunologic therapy. However, the immune milieu is impaired after autoSCT. We hypothesized

that autologous lymphocyte infusion would restore immune competence, allowing

immunotherapies such as cancer vaccines to elicit tumor antigen–specific immunity in the setting

of autoSCT. In this pilot study (NCT01380145), we investigated safety, immunologic, and

clinical outcomes of autologous lymphocyte infusion combined with peri-autoSCT

immunotherapy with recombinant MAGE-A3 (a multiple myeloma–associated antigen) and

adjuvant. Thirteen multiple myeloma patients undergoing autoSCT were enrolled. Autologous

lymphocyte infusion and MAGE vaccination were well tolerated. Combination immunotherapy

resulted in high-titer humoral immunity and robust, antigen-specific CD4+ T-cell responses in all

subjects, and the responses persisted at least one year post-autoSCT. CD4+ T cells were

polyfunctional and Th1-biased. CD8+ T-cell responses were elicited in 3/13 subjects. These cells

recognized naturally processed MAGE-A3 antigen. Median progression-free survival was 27

months, and median overall survival was not reached, suggesting no differences from standard-

of-care. In 4/8 subjects tested, MAGE-A protein expression was not detected by

immunohistochemistry in multiple myeloma cells at relapse, suggesting therapy-induced

immunologic selection against antigen-expressing clones. These results demonstrated that

autologous lymphocyte infusion augmentation of autoSCT confers a favorable milieu for

immunotherapies such as tumor vaccines. This strategy does not require ex vivo manipulation of

autologous lymphocyte products and is an applicable platform for further investigation into

combination immunotherapies to treat multiple myeloma.

Introduction

Multiple myeloma, a malignancy of immunoglobulin (Ig)-secreting plasma cells, is the

second most common hematologic cancer (1). The introduction of agents such as

immunomodulatory drugs (ImiDs) and proteasome inhibitors and use of autologous stem cell

transplantation (autoSCT) as standard consolidation therapy have improved both disease-free and

overall survival. Despite these advances, relapse is the rule and most patients die of their disease.

In contrast, allogeneic (allo)SCT studies with long-term follow-up demonstrated a survival

plateau in multiple myeloma, indicating a subset of patients who are cured (2). Depletion of T

cells from allogeneic grafts increase relapse rates, and objective responses are observed with

donor lymphocyte infusion (3), demonstrating that immunologic therapy, in the form of T cell-

mediated “graft-versus-myeloma” effect can confer long-term remission. However, alloSCT is

only applicable in a minority of multiple myeloma patients due to age and health restrictions and

to procedure-related morbidity and mortality. Broadly applicable strategies that confer safe and

effective “host-vs-myeloma” immune responses are therefore needed.

The period around autoSCT invites exploration of additional multiple myeloma

immunotherapies, as autoSCT generally confers the lowest tumor burden in the natural history of

the disease and engraftment of the cellular immune compartments provides the opportunity to

sculpt anti-tumor immunity. Challenges to this approach include delayed immune reconstitution,

which results in poor responses to infectious disease vaccines (4). Rapoport and colleagues

demonstrated that pre-autoSCT vaccination followed by early (day +12) post-autoSCT transfer

of vaccine-primed T cells, activated ex vivo with anti-CD3/anti-CD28–coated beads, and post-

transplant booster vaccinations restored immune competence to a pneumococcal vaccine in

patients undergoing autoSCT (5). Patients who did not receive T-cell transfer until day +90

failed to develop durable pneumococcal immunity, demonstrating the need for early T-cell

reconstitution during the post-SCT period (5). Follow-up studies have expanded on this use of

cellular augmentation modalities for autoSCT as immunotherapy strategies for multiple

myeloma, demonstrating generation of immune responses to infectious and tumor antigens, as

well as the safety of even earlier adoptive T-cell transfer (day +2 or 3 post-SCT)(6,7). Several

groups have explored analogous adoptive transfer strategies following autoSCT, including the

use of activated marrow-infiltrating lymphocytes (8), or T cells genetically modified to express

transgenic T-cell (9) or chimeric antigen (10) receptors. Although these studies have

demonstrated the feasibility and safety of these approaches, the use of ex vivo-activated T cells

has some disadvantages, including risk of autologous graft-versus-host-disease (GVHD)(11) and

need for extensive manipulation in a specialized cell-processing facility, which adds expense and

complexity and limits these approaches to a few centers. Simpler reconstitution strategies could

be applied broadly at any transplant center. Murine studies demonstrated that infusion of

unmanipulated, vaccine-primed lymphocytes within a few days of myeloablative therapy

followed by post-transfer vaccination leads to robust in vivo expansion of transferred cells and

long-lasting anti-tumor immunity (12).

Based on these results, we hypothesized that augmentation of autoSCT with vaccine-

primed autologous lymphocyte infusion would create a favorable milieu for the generation of

tumor antigen-specific immune responses. We conducted a pilot study of this strategy with an

immunotherapeutic product containing recombinant Melanoma Antigen Gene A3 (recMAGE-

A3) protein, a member of the Cancer-Testis Antigen (CTAg) group of cancer-associated genes

that has been investigated in tumor immunotherapy (13). MAGE-A3 is expressed in 30-40% of

newly diagnosed multiple myeloma patients and up to 77% of relapsed multiple myeloma

patients (14-18), is immunogenic in multiple myeloma patients (19,20), and is a functional target

that inhibits apoptosis in multiple myeloma cells (21). In our study, we administered recMAGE-

A3 with AS15 immunostimulant (22) to 13 multiple myeloma subjects pre- and post-autoSCT in

conjunction with early post-SCT vaccine-primed autologous lymphocyte infusion. We report

here analysis of safety, clinical outcomes, and immune responses. This strategy was safe in the

target multiple myeloma population and supported robust MAGE-A3-specific humoral and CD4+

T-cell immune responses. These results demonstrate that autologous lymphocyte infusion-

augmented autoSCT broadens the scope of immunotherapeutic strategies to treat multiple

myeloma.

Materials and Methods

Subjects: Subjects with symptomatic multiple myeloma, who were within 12 months of starting

treatment, had achieved a Very Good Partial Response (VGPR) or better by International

Myeloma Working Group (IMWG) criteria after induction therapy (23), had no prior stem cell

transplant, and were eligible to receive high dose chemotherapy and autologous stem cell rescue

by standard institutional criteria, were enrolled at three centers (see Supplementary Fig. S1 for

full eligibility criteria). Choice of induction therapy was not dictated by the study. Subjects

must have had a bone marrow biopsy specimen or plasmacytoma that was positive for MAGE-A

antigen by immunohistochemistry (IHC). Written informed consent was obtained from each

subject and the study was conducted with approval of each institution’s IRB in concordance with

the Declaration of Helsinki. The trial was registered in clinicaltrials.gov under NCT01380145.

Immunohistochemistry: IHC was performed on formalin-fixed, paraffin-embedded bone marrow

or plasmacytoma biopsy specimens using the M3H67 monoclonal antibody (mAb) specific for

MAGE-A3, as well as other homologous MAGE-A family members, as described (14,16). The

following scoring system was used: negative: focal: <5% multiple myeloma cells positive; +:

5-25%; ++: 25-50%; +++: 50-75%; ++++: >75%. Subjects with a score of + to ++++ were

deemed “MAGE-A positive” and were eligible for the study. When feasible, IHC for MAGE-A

expression was also performed at time of progression.

Vaccine: The vaccine preparation was comprised of 300 mcg of the recombinant ProtD-MAGE-

A3/His, a 432-amino acid fusion protein containing 109 amino acids of Haemophilus influenzae

B Protein D (ProtD), the full-length MAGE-A3 protein, and a polyhistidine tail (His), and the

proprietary immunostimulant AS15. AS15 is a combination of 3-O-desacyl-4’- monophosphoryl

lipid A (MPL, 50 µg, produced by GSK), Quillaja saponaria Molina, fraction 21 (QS-21, 50 µg,

licensed by GSK from Antigenics LLC, a wholly owned subsidiary of Agenus Inc., a Delaware,

USA corporation) and CpG 7909 synthetic oligodeoxynucleotides (ODNs) containing

unmethylated CpG motifs (420 µg), in a liposomal formulation (22). Each dose was given

intramuscularly.

Treatment regimen (Supplementary Fig. S1): Injection #1 was given 6-7 weeks before

autologous stem cell transplantation (auto-SCT), at least three weeks after completing induction

therapy. Three weeks later subjects had steady-state leukapheresis to collect and freeze vaccine-

primed PBL, with a goal of 1 x 108 CD3+ cells/kg, followed by stem cell mobilization per

institutional standards. About 1 week after mobilization, subjects received high dose melphalan

(200 mg/m2), followed by stem cell infusion on day 0 and autologous lymphocyte infusion on

day +3. Immunizations #2 to 6 were given every three weeks (+/- 3 days) starting on day 10

post-SCT (i.e. days 10, 31, 52, 73, 94). Two additional immunizations (#7 and 8) were given at

3-month intervals (+/- 7 days) (i.e. days 180 and 270). Maintenance lenalidomide was allowed at

the discretion of the treating physician, starting 3 months or more after autoSCT.

Sample collection: Blood samples for immunologic correlates were obtained pre-treatment, 3

weeks after immunization #1 (at time of PBL leukopheresis), at time of stem cell collection, and

post-SCT on day 31 (immunization #3), day 73 (immunization #5), day 194 (2 weeks post-

immunization #7), day 284 (2 weeks post-immunization #8) and day 365 (1 year post-SCT).

Bone marrow aspirate samples were collected pre-treatment, and at 3 months and 1 year post-

SCT. Plasma was collected by centrifugation of whole blood and cryopreserved. PBMCs were

isolated by Ficoll-Paque gradient and cryopreserved for immunophenotyping and cellular assays.

Assessment of Humoral Immunity: Antibodies to MAGE-A3 were measured by ELISA, as

previously described (24), using two different preparations of MAGE-A3: a baculovirus-

produced, histidine-tagged recombinant protein, and a pool of 20mer overlapping peptides

covering the entire sequence of MAGE-A3 (named MAGE-A3 1-30). Briefly, peripheral blood

and bone marrow plasma was thawed and tested in duplicate for each patient in four-fold serial

dilutions from 1/100 to 1/100,000 for presence of IgG against the two different preparations of

MAGE-A3. These antigen preparations were chosen to confirm specificity to MAGE-A3, by

avoiding detection of potential microbial components related to the vaccine product (which was

E. coli produced) and by measuring reactivity to synthetic linear epitopes. All patients were

tested at selected time points against a “tagless” MAGE-A3 protein produced in E. coli but

lacking the histidine tag, and some patients were also tested against a different pool of 57 shorter

MAGE-A3 peptides provided by GSK (named MAGE-A3 1-57). Reactivity to Protein D was

assessed using an E. coli-produced protein provided by GSK. Results are expressed as reciprocal

titers based on the predicted dilution at which a linear extrapolation of the titration curve meets a

cutoff determined from a healthy donor serum pool. Titers were considered significant if >100.

Induction of responses was considered significant if going from undetectable (<100) to

detectable (>100) or if increasing in titers over time by at least 4x. Titers at individual time

points were also confirmed side-by-side for all patients in the same assay, to allow cross-patient

comparisons.

Assessment of Cellular Immunity: Monitoring of IFN-producing CD4+ and CD8+ T cells

specific for MAGE-A3 was performed by ELISPOT and intracellular cytokine staining

following in vitro 11-day (for CD8) or 20-day (for CD4) pre-sensitization as previously

described (24). Briefly, CD4+ and CD8+ T cells were sequentially purified from PBMCs using

magnetic beads (Life Sciences) and stimulated once with remaining irradiated CD8–CD4– cells

pulsed overnight with pools of overlapping 20mer peptides covering the entire MAGE-A3

sequence (30 peptides in total overlapping by 10AA). As controls, an overlapping peptide pool

covering the entire sequence of influenza A nucleoprotein (NP) was used. After a culture period

of 11 days for CD8+ T cells and 20 days for CD4+ T cells in RPMI containing 10% SAB

supplemented with glutamine, antibiotics, non-essential amino acids, IL2 (10U/ml; Roche, Basel,

Switzerland), and IL7 (20ng/ml, R&D Systems, Minneapolis, MN), CD8+ and CD4+ T cells were

harvested and tested by IFN- ELISPOT on autologous EBV-B lymphoblastoid cells (B-EBV) or

PHA-activated T cells (T-APC) pulsed with MAGE-A3 peptide pools (1-30 20mers or 1-57

shorter peptide pool), control NP peptide pool, or irrelevant DMSO. Direct ex vivo responses

(using PBMC without pre-sensitization) were also assessed by ELISPOT at selected time points.

B-EBV cells were infected overnight with adenovirus or vaccinia virus recombinant for MAGE-

A3 as cognate target or NY-ESO-1 as control.

ELISPOT: Flat-bottomed, 96-well nitrocellulose plates (MultiScreen®-HA; Millipore, Bedford,

MA) were coated with IFNγ mAb (4 µg/ml, 1-D1K; Mabtech, Stockholm, Sweden) and

incubated overnight at 4°C. After washing with RPMI, plates were blocked with 10% human AB

type serum for 2 h at 37°C. Pre-sensitized CD8 or CD4 T cells (5x104 and 1x104) and 5x104

target EBV-B or T-APC cells pulsed overnight with 1µM peptides from MAGE-A3 or control

peptides were added to each well and incubated for 20 h in RPMI medium 1640 without serum.

PMA/Ionomycin was used for quality control of CD8 and CD4 T cells (1x104 without targets).

Plates were then washed thoroughly with water containing 0.05% Tween 20 to remove cells, and

IFN-γ mAb (0.2 µg/ml, 7-B6-1-biotin; Mabtech) was added to each well. After incubation for 2

h at 37°C, plates were washed and developed with streptavidin-alkaline phosphatase (1 µg/ml;

Roche) for 1 h at room temperature. After washing, substrate (5-bromo-4-chloro-3-indolyl

phosphate/nitroblue tetrazolium; Sigma) was added and incubated for 10 min. After final washes,

plate membranes display dark-violet spots representing IFN- secreting CD8+ or CD4+ T cells

that were counted with the CTL Immunospot analyzer and software (Cellular Technologies,

Cleveland, OH). The average from duplicates was calculated, and a positive response was

determined as >50 spots and >2x number of spots for control targets pulsed with DMSO. Results

are shown after subtraction of spots with DMSO control targets and elevated spot counts to

cognate antigen-pulsed targets not reaching the threshold for positivity due to high reactivity

with DMSO-pulsed targets were assigned a value ≤50. An immunological response was defined

as MAGE-A3 CD4 or CD8 T cells going from undetectable at baseline (<50 spots) to detectable

post immunization (see above) against MAGE-A3 peptides compared to negative control

peptides. In case of preexisting MAGE-A3 CD4+ or CD8+ T cells, an immunological response

was defined as at least 4x increase in number of specific spots over baseline.

Ex vivo responses: Briefly, ex vivo responses were measured at selected time points using a 48hr

IFN- ELISPOT after thawing PBMC and overnight rest, using direct pulsing of 1 million

PBMCs (no separate antigen presenting cells) with a pool of MAGE-A3 overlapping 20mer

peptides (1-30) or with control DMSO or Influenza NP peptide pool. PMA/Ionomycin was used

as a positive control. Ex vivo responses to MAGE-A3 were considered positive if >25 spots and

at least 2x the levels observed to negative control DMSO.

Intracellular staining of cytokines: B-EBV target cells pulsed with shorter MAGE-A3 peptide

pool 1-57, NP pool, or DMSO were washed and incubated with MAGE-A3 1-30 or NP pre-

sensitized effector T cells at a 1: 2 ratio in 200 µl X-VIVO-15 at 37 ºC for 6 hours. Brefeldin-A

(Sigma-Aldrich, St. Louis, MO) at 10 g/ml was added after the first two hours of culture. Cells

were then fixed and permeabilized (BD Biosciences, San Jose, CA), and stained with appropriate

antibodies against CD4, CD8, IFN, TNF, IL4, or IL5 (BD Biosciences). PMA/Ionomycin was

used as a positive control. Cells were subsequently analyzed by flow cytometry with gating on

morphologically defined lymphocytes, CD4- or CD8-positive cells. A positive response was

determined as >0.5% and >2x the percentage for DMSO control target. An immunological

response was defined as MAGE-A3 CD4+ or CD8+ T cells going from undetectable at baseline

(<0.5%) to detectable post immunization (see above) against MAGE-A3 peptides compared to

negative control peptides. In case of preexisting MAGE-A3 CD4+ or CD8+ T cells, an

immunological response is defined as at least 4x increase in percentage of MAGE-A3 specific T

cells over baseline. Results are shown after subtraction of percentage with DMSO control targets

and elevated percentages to cognate antigen-pulsed targets not reaching the threshold for

positivity due to high reactivity with DMSO-pulsed targets were assigned a value ≤0.5%.

Generation of CD4+ T-cell lines: Pre-sensitized CD4+ cells were re-stimulated with MAGE-A3

overlapping peptide-pulsed autologous antigen-presenting cells (APCs), and then CD154+CD4+

cells (representing the activated MAGE-A3-specific fraction) were sorted by flow cytometry.

This CD154+ fraction then underwent serial non-specific expansions over 3-5 weeks using PHA,

IL2, IL7, and irradiated feeder cells (allo-PBMC), as described (25), to generate CD4+ T-cell

lines. Lines were then tested for MAGE-A3 specificity and IFN production via ELISPOT.

Cytotoxicity against MAGE-A3-pulsed or control autologous target cells was assessed using

europium-release assay according to manufacturer’s instruction (Delfia, Perkin-Elmer).

Clinical outcomes: Adverse events were graded according to NCI CTCAE v4. Hematologic

responses were determined according to IMWG criteria. Progression-free survival (PFS) and

overall survival (OS) was calculated from time of enrollment and estimated using the Kaplan-

Meier method (Graphpad Prism 5.0). Data cut-off date for PFS and OS was 06/30/2015.

Statistical analyses: The primary study endpoint was safety and tolerability; secondary endpoints

were immune responses and clinical outcomes. Frequency of adverse events and hematologic

responses were analyzed descriptively. Changes in antibody titers and ELISPOT over time from

baseline were assessed using linear mixed effects models with random subject intercept.(26). An

immune response was defined as (1) increase in antibody titers in at least two post-treatment

samples of at least 5-fold over baseline, or (2) increase in T-cell frequency in at least two post-

treatment samples of at least 5-fold over baseline and 3-fold over background. This was designed

as a pilot study to enroll up to 16 subjects to obtain at least 12 “evaluable” for immune response,

defined as having completed pre-SCT vaccination, PBL transfer and at least 4 out of the first 5

post-SCT vaccinations. At the time this “evaluable” threshold was met for the 12th subject, a 13th

subject had already been enrolled, so a total of 13 subjects were treated on study.

Results

Subject characteristics

Ninety-three subjects were pre-screened over 18 months. Thirty (32%) were MAGE-A3 positive,

in agreement with previously reported frequencies of expression in newly diagnosed subjects.

Thirteen subjects were enrolled (see Supplementary Fig. S1 for subject flow chart). Subject

characteristics are listed in Table 1. Median age was 56, and 5/11 tested had at least 1 high-risk

cytogenetic abnormality, which is higher than expected for the general multiple myeloma

population. Twelve of 13 subjects had lenalidomide and bortezomib during induction, and 9/13

had a change in induction therapy prior to enrollment due to suboptimal response, indicative of

high-risk clinical behavior regardless of cytogenetics/FISH. Twelve subjects were in VGPR

entering the study, with 1 in complete response (CR).

Feasibility and safety

PBL collection for autologous lymphocyte infusion yielded a median of 1.4 x 108 CD3+ cells/kg

(range 1.0-2.6), with all subjects reaching the targeted goal. Pre-SCT immunization did not

impact stem cell yield, with a median of 11.4 x 106 CD34+ cells/kg (range 8.3 – 37.2) collected.

Autologous lymphocyte infusions were well tolerated, and no engraftment-like syndrome or

autologous GVHD was noted. Engraftment to an absolute neutrophil count (ANC) > 500/µl

occurred at a median of 11 days. Protocol treatment was feasible with 12/13 subjects completing

the planned treatment, with only one missed vaccination, and one subject withdrawing early due

to progression. There were no unexpected autoSCT-related toxicities and no treatment-related

deaths. Treatment-related-adverse events were primarily Grade 1 and self-limited, with the most

common being injection site reaction/pain reported in 54% of subjects (Supplementary Table

S1). There were eight SAEs reported in four subjects (Supplementary Table S2), three of which

were possibly related to protocol treatment. One subject developed self-limited grade 2 myalgia

and grade 1 injection site-reaction shortly after vaccination which led to a 24-hour

hospitalization. One subject developed a skin disorder 14 months after the final vaccination,

initially diagnosed as psoriasis, later evolving to grade 3 mixed connective tissue disorder. No

other autoimmune toxicities were noted. Lenalidomide maintenance was discontinued in one of

five subjects due to grade 1 rash, which is a common AE for this agent, and there were no

unexpected toxicities associated with lenalidomide.

Clinical outcomes

At enrollment, 12 subjects were in VGPR and one in CR after induction therapy. Restaging at

three months post-SCT showed six subjects in CR (three in stringent CR (sCR)) and seven

subjects in VGPR. Lenalidomide maintenance was started in five subjects, though discontinued

in two subjects within two months of initiation (one for progression, one for rash). At the one-

year post-SCT restaging, there were four VGPR and five CR (four in sCR), including two

subjects whose hematologic response improved between three and 12 months post-autoSCT,

neither of whom received lenalidomide maintenance. Four subjects had progression at or before

the one-year restaging visit. With a median follow-up of 31 months, three subjects remain

progression-free, and median PFS is 27 months. Three subjects have died, two from myeloma

and one from complications of salvage allogeneic transplant, with median OS not reached (Fig.

1). Understanding the limited statistical power of study, we observed no trend suggesting

association between PFS and degree of MAGE-A expression at diagnosis or lenalidomide

maintenance vs no maintenance. No association was observed between PFS and the immune

response data described in the following sections, including spreading of humoral immune

response, MAGE-A3-specific CD8+ T-cell response, or loss of MAGE-A3 expression.

Humoral Immune Responses

All subjects developed robust MAGE-A3-specific antibody titers in response to protocol therapy.

High antibody titers (1:104-106) against the immunized antigen were detected as early as three

weeks after the first post-priming vaccine and persisted to at least 1-year post-ASCT (Fig. 2A).

Titers from peripheral blood and bone marrow plasma were concordant (Supplementary Fig. S2).

Kinetics of antibody induction differed from subject to subject. Three subjects seroconverted

after a single immunization (pre-SCT; M15, M27, and S25). Another eight subjects

seroconverted by day 31 after autoSCT, following two immunizations (pre-SCT and Day 10

post-SCT; F06, F10, M08, M26, S02, S10, S13, S36). The remaining two subjects seroconverted

by day 73, after 4 immunizations (F02, S33). All antibody responses were significantly higher

than baseline (p<0.001 at each time point). Specificity of antibody responses to MAGE-A3 was

confirmed in all subjects using a pool of overlapping 20-mer peptides covering the full-length

protein (Fig. 2B) or tagless MAGE-A3 protein (Supplementary Table S3), though recognition of

linear epitopes (peptide pool) was weaker and sometimes delayed compared to full-length

protein recognition. Epitope mapping demonstrated that vaccination induced a polyclonal

response against at least 7 distinct epitope regions of the MAGE-A3 protein (Fig. 2C). The

diversity of responses agreed with observations from a prior study (LUD99-010) in NSCLC

using recombinant MAGE-A3 with an immunostimulant composed of MPL and QS21 in

oil/water emulsion that did not contain CpG 7909 (27). However, average titers in the current

study were 1-2 logs higher than those in the previous study, illustrating the potency of the CpG-

containing adjuvant (24,27). Immunoglobulin isotype and IgG subclass analysis analyses showed

that IgG was the dominant isotype, with IgG1 and IgG3 being the dominant subclasses (Fig 2D).

High-titer antibody responses to the H. influenzae Protein D component of the vaccine were also

seen in all subjects after treatment (Fig. 2E). Antibody responses against a panel of control

proteins not included in the vaccine did not change significantly in the majority of subjects,

although three subjects demonstrated sustained titers to four non-immunized, cancer-associated

antigens after vaccination, raising the possibility of antigen spreading in a minority of cases

(Supplementary Fig. S3).

Cellular Immune Responses

All subjects demonstrated induction and/or expansion of CD4+ T-cell responses against MAGE-

A3, which persisted to at least 1-year post-SCT as assessed by in vitro re-stimulation assay (Fig.

3A). Seven subjects had no baseline reactivity but developed MAGE-A3-specific CD4+ T cells

after the first (F06, M26, S02), or second vaccination (F02, F10, M08, M27), mirroring what was

observed with antibody titers. Six subjects (M15, S10, S13, S25, S33, S36) had baseline CD4+ T-

cell responses that were detected by co-incubation with peptide-loaded antigen-presenting cells

(APC), either autologous T-cell APC (T-APC) or B cell EBV blasts (B-EBV). All of these

subjects showed significant increases in CD4+ T-cell responses after vaccination plus autologous

lymphocyte infusion (Fig. 3B, mean at baseline: 229 spots, mean from d31 onward: 1155 to 2295

spots, p<0.001 at each time point). Given the expansion of MAGE-A3-specific CD4+ T cells

after in vitro re-stimulation, we also examined direct ex vivo cellular responses without in vitro

re-stimulation and found that 5 of 13 subjects had detectable ex vivo ELISPOT responses against

MAGE-A3, typically at later time points after receiving the final 2 booster vaccinations (Fig.

3C). Once detected, CD4+ T-cell responses were consistent over time and were generally of

similar magnitude regardless of APC or peptide pool used (20mers vs. shorter peptides,

Supplementary Fig. S4). As a control, influenza nucleoprotein (NP)-reactive CD4+ T cells were

detected throughout treatment in most subjects and on average did not significantly change over

time (Fig. 3B).

Cytokine concentrations were quantified in the supernatants of ELIspot co-cultures,

demonstrating Ag-specific secretion of GM-CSF, TNFα, MIP-1β, IL5, and IL8, whereas IL2,

IL19, and IL10 were not detected (Fig 3D). Intracellular cytokine staining showed that vaccine-

elicited, MAGE-A3-specific CD4+ T cells produced both IFNγ and TNF but not IL4, indicating

polyfunctional, Th1-biased immune responses (Fig. 3E).

In order to further characterize the functional capacity of these vaccine-induced CD4+ T

cells, we sorted and enriched MAGE-A3-specific CD4+ cells from PBMC of 3 subjects obtained

at day +194 post-autoSCT, based on CD154 (CD40L) expression following antigen stimulation

(Fig. 3F). All of these T-cell populations showed specific IFNγ production upon re-exposure to

autologous APC pulsed with MAGE-A3 overlapping peptides and full-length protein compared

to negative control stimulation (Fig. 3G), and CD4+ cells from subjects S13 and S25 also

recognized autologous target cells transduced by viral vectors expressing full-length MAGE-A3.

This demonstrates the capacity of the CD4+ cells to recognize endogenously-processed and

presented epitopes that mimicked the natural state of a tumor expressing intracellular MAGE-A3

and HLA class II. We did not observe direct cytolytic activity by these CD4+ T cells against

MAGE-A3-expressing targets.

In contrast to the activation of Ag-specific CD4+ T cells, CD8+ T-cell responses were

noted in only three subjects (F06, M27, S25, Fig. 4A), in agreement with results from previous

vaccines with full-length recombinant proteins favoring CD4+ over CD8+ T-cell induction

(28,29). These CD8+ cells were capable of recognizing naturally-processed MAGE-A3, as

demonstrated by IFNγ production against targets transduced by viral vectors expressing full-

length MAGE-A3 (Fig. 3C). All three of these subjects had detectable ex vivo CD4+ responses

(Fig. 4B). Post-autoSCT CD8+ responses against influenza NP were seen in 10/13 subjects,

demonstrating that CD8+ function was not globally impaired in most subjects (Fig. 3B). Eight

subjects who relapsed had bone marrow or plasmacytoma biopsies available; in contrast to their

original tumor (Fig. 4C, D), four (50%) no longer had detectable MAGE-A+ multiple myeloma

cells by immunohistochemistry (Fig. 4E, F). Loss of MAGE expression has not been reported in

longitudinal studies in multiple myeloma, suggesting that the experimental therapy exerted

selective pressure against MAGE-A3+ clones. However, there was no correlation between CD8+

T-cell activity and loss of MAGE-A3 expression at progression.

Immunophenotyping of peripheral blood mononuclear cells by time-of-flight mass

cytometry (CyTOF) demonstrated a significant increase in effector and central memory CD4+ T

cells (CD45RA-, CCR7–, Fig. 5A), together with significantly higher expression of co-

stimulatory molecules 4-1BB (Fig. 5B) and ICOS (Fig. 5C), the activation marker CD38 (Fig.

5D), the immune checkpoint receptors PD-1 (Fig. 5E), but not LAG-3 (Fig. 5F) on CD4+ T cells

in the post-autoSCT period. Cell were sustained through the study period, indicating a higher

state of activation in the post-ASCT period compared to baseline.

In summary, autologous lymphocyte infusion in combination with autoSCT is a safe and

feasible platform for immunotherapy, including therapeutic tumor vaccines, for multiple

myeloma. Twelve of 13 subjects completed the entire protocol, with one coming off early for

progression. This strategy provided a favorable immune milieu for antigen-specific humoral and

CD4+ T-cell immune responses in all subjects. CD8+ immunity was observed in three subjects.

Loss of MAGE-A3 expression at progression in 4/8 of evaluated subjects indicates immunologic

efficacy of this approach. MAGE-A3-specific humoral and cellular responses are summarized in

Supplementary Fig. S5.

Discussion

The results reported here provide proof-of-principle that autologous lymphocyte infusion

augmentation of autoSCT is a promising component for multiple myeloma combination

immunotherapy. This strategy is safe, feasible, and did not adversely impact the delivery of

standard-of-care autoSCT. Pre-clinical studies demonstrate that the lymphopenic period after

myeloablative chemotherapy and autoSCT is a favorable environment for generating anti-tumor

immunity through vaccination. Several mechanisms have been identified, including 1) tumor

antigen release due to cell death; 2) enhanced dendritic cell activation due to greater availability

of Toll-like receptor agonists from translocation of gut flora and other microbiota; 3) increased

availability of T-cell survival factors (e.g. IL7 and IL15); and 4) elimination of suppressor

populations (e.g. myeloid-derived suppressor cells (MDSC) and Tregs) (30-33). The addition of

autologous lymphocyte infusion, in this case in conjunction with therapeutic tumor vaccinations,

resulted in robust, humoral and Th1-biased CD4+ T-cell immunity in all subjects, and CD8+ T-

cell activity in three subjects. These results support further investigation into combined

immunotherapy building upon the platform of autologous lymphocyte infusion-augmented

autoSCT, since evidence is accumulating for a critical role for the immune system in controlling

this disease. Both adaptive and innate immune responses against autologous multiple myeloma

cells were detected in MGUS and smoldering multiple myeloma subjects but were lost with

progression to active disease. These responses could be restored ex vivo with appropriate

stimulation (34,35). Cellular and humoral immune responses against myeloma-associated

antigens were detected in multiple myeloma subjects after undergoing alloSCT, and were

correlated with clinical outcome (20). In the same study, 22 subjects who underwent autoSCT

were screened for humoral immune responses to CTAgs, and only one subject developed an

antibody response against MAGE-A3 after transplant, suggesting that autoSCT alone is not

sufficient to induce specific immune responses against MAGE-A3 in the majority of patients.

The strategy of pre-SCT immunization and lymphocyte collection, early post-SCT autologous

lymphocyte infusion, and booster immunizations was designed to leverage the favorable

immunological milieu following myeloablative therapy and the diversity of mature, competent

lymphocytes provided by autologous lymphocyte infusion, without incurring the time, resources,

and expense of ex vivo engineering or manipulation of T cells. These strategies may be broadly

applicable, as autoSCT is already standard-of-care management in eligible multiple myeloma

subjects and the methods and resources described are available at most transplant centers.

The combined vaccine and autologous lymphocyte infusion augmentation strategy was

effective in stimulating humoral and Th1-biased CD4+ T-cell responses to the immunized

antigen, with 100% of subjects responding. These results indicate that the vaccine antigen was

efficiently taken up and processed by antigen-presenting cells and presented to CD4+ T cells on

MHC class II, and that the AS15 immunostimulant formulation promoted Th1-biased

priming/boosting. This occurred despite the immune compromise exhibited by myeloma subjects

in general and especially after autoSCT, indicating the power of this strategy to generate antigen-

specific immunity in the autoSCT setting.

Analysis of MAGE-A3 expression in multiple myeloma cells at diagnosis and relapse

demonstrated that four of eight subjects did not express MAGE-A3 by IHC in their multiple

myeloma cells at the time of progression. MAGE-A3 expression is typically more frequent in

relapsed multiple myeloma compared to newly diagnosed subjects (14). In a prior longitudinal

analysis, MAGE-A3 expression was sustained over time (18), and there are no published

examples of MAGE-A3 expression being lost from diagnosis to relapse. These results suggest

that our treatment strategy exerted immunologic pressure on multiple myeloma cells, either by

eliminating MAGE-A3-expressing clones and/or inducing loss of MAGE-A3 expression as a

means of escape despite the absence of broad CD8+ cell activity, reminiscent of clinical reports

describing adoptive transfer of tumor Ag-specific CD4+ clones inducing regression of tumors

even in the absence of cytolytic activity (36-38). This may occur through both direct killing of

targets, or through indirect mechanisms such as cytokine production that recruits macrophages,

NK cells, or other effector cells into the microenvironment (19,37,39,40). In addition,

downregulation of tumor antigen expression has been described following T-cell therapy

targeting CTAg (9,41). The four subjects who progressed with continued MAGE-A3 expression

may have had inadequate CD8+ T-cell responses, down-regulation of MHC class I and/or II on

tumor cells, inhibitory Treg activity, and/or engagement of inhibitory immune co-receptors such

as PD-L1, which is commonly expressed on multiple myeloma cells and in the tumor

microenvironment (42,43). In our study, CD4+ T cells post-SCT demonstrated elevated PD-1

expression, raising the intriguing possibility that addition of an immune checkpoint inhibitor

targeting the PD-1/PD-L1 axis may increase the clinical efficacy of this strategy, as has been

demonstrated in pre-clinical and ex vivo studies (44,45).

This study was not powered for assessment of clinical efficacy and we did not observe a

trend towards benefit over standard-of-care autoSCT in this treatment group despite the

induction of humoral and CD4+ cellular immunity. Several factors may have impacted the

clinical outcome. First, there was little induction of CD8+ T-cell activity. This is similar to

another study using the same recMAGE-A3+AS15 formulation in melanoma subjects, where

CD4+ T-cell responses were seen in 76% of subjects and CD8+ T-cell responses in only 3% (46).

This may have also contributed to the limited clinical impact of this vaccine formulation in phase

3 studies in NSCLC and melanoma (47). Second, immunity to a single tumor-associated Ag may

not be sufficient to control disease. Myeloma is characterized by clonal diversity and evolution

(48,49). Therefore, elimination of a subset of MAGE-A3-expressing clones, as in our study, may

not be sufficient to induce lasting remission as other clones will expand and progress. Finally,

MAGE-A3 itself may be a malignancy factor that contributed to poor clinical outcome, as

expression of this class of genes confers a survival advantage in multiple myeloma cells (14,50)

and was associated with advanced stage and poor prognosis (18,51,52). Expression of a cluster

of CTAg, including MAGE-A3, was associated with resistance to CTLA-4 blockade in

melanoma; the authors speculated that the inhibition of immunogenic cell death mediated by

these genes may negatively impact T-cell priming to tumor-associated antigens (53). Similar

mechanisms may have contributed to poor CD8+ T-cell priming observed in this study.

Therefore, a median PFS of 27 months in this MAGE-A3+ cohort, 45% of which had high-risk

cytogenetics and most of whom received no maintenance therapy, is consistent with PFS

reported in other post-SCT cellular therapy trials in the high-risk multiple myeloma population

(6,8,9).

In conclusion, we have demonstrated that autologous lymphocyte infusion and

therapeutic tumor vaccine augmentation of autoSCT is safe, feasible, and stimulates tumor Ag-

specific B and Th1-biased CD4+ T-cell immunity. Clinical outcome and modest CD8+ T-cell

activity suggest that the vaccine formulation used here was not sufficient to confer long-term

disease control. However, the immunologic efficacy of this strategy supports further

investigation of other combinations of immunotherapy built upon this platform. Future clinical

trials may test vaccines that incorporate a diversity of myeloma-associated antigens utilizing

synthetic long peptides to better promote CD8+ T-cell priming (54), along with potent

immunostimulants such as AS15 to ensure integrated CD4+ and B cell immunity. Another

promising approach under investigation uses individualized neoantigen vaccines composed of

peptides containing non-synonymous, immunogenic epitopes derived from whole exome

sequencing of tumor specimens (55). Immunomodulatory drugs (ImiDs) such as lenalidomide

are approved for multiple myeloma and have a plethora of favorable immunologic activities,

including T, B, and NK cell activation, dendritic cell maturation, and promotion of antigen

presentation (56). Immune checkpoint inhibitors targeting the B7/CTLA-4 and PD-1/PD-L1 axes

are approved for solid tumor indications and are under investigation in multiple myeloma. The

combination of these strategies with vaccine and autologous lymphocyte infusion augmentation

of autoSCT in rational sequence and composition may realize the goal of “host vs myeloma”

immunity that recapitulates the alloSCT experience in a therapy that is feasible for the majority

of multiple myeloma patients. These strategies may also be applicable to other hematologic

malignancies where autoSCT is a common salvage therapy, such as Hodgkin’s and non-

Hodgkin’s lymphomas.

Acknowledgments: The authors thank the patients and family members for their participation in the study, as well as Linda Thibodeau and Stephanie Rosati at Fox Chase Cancer Center; Linda Pan and Stephane Bertolini at Ludwig Institute for Cancer Research; the Cancer Research Institute; and Adolfo Aleman, Michael Donovan and Luis Ferran at Icahn School of Medicine at Mt. Sinai. They also thank Rafik Fellague-Chebra, Olivier Gruselle, Peter Van den Steen, Sophie Timmery and the GSK Vaccines team for study support and critical review of the manuscript. Funding: The study was sponsored by the Ludwig Institute for Cancer Research (NY, NY), with funding support from GlaxoSmithKline Biologicals SA. S.G. acknowledges funding support for immunological monitoring from the Cancer Research Institute. Author contributions: Contributions: ADC, NL, SG and HJC designed the study, performed research, analyzed data, and wrote the manuscript. SN, NI, AAJ, and KA performed research and analyzed data. IT, AR, AHCM, HS, KZ, and SK collected, assembled and entered data. LP, SB, and RV assembled and analyzed data. All authors approved the manuscript. Competing interests: A.D.C. received commercial clinical trial support from GlaxoSmithKline, commercial research support from Novartis and Bristol-Myers Squibb, and compensation for consulting/advisory boards from Bristol-Meyers Squibb, GlaxoSmithKline, Celgene, Seattle Genetics, Oncopeptides, Kite Pharma, Takeda, Janssen, and Array Biopharma. N.L. received commercial research support from GlaxoSmithKline, Takeda, Karyopharm, Sanofi, Amgen, Pharmacyclics, and Array Biopharma, and compensation for consulting/advisory boards Karyopharm and Amgen. S.G. received commercial research support from Janssen R&D, Bristol-Myers Squibb, Pfizer, and Immune Design, and compensation for consulting/advisory boards from Merck, OncoMed, B4CC, and Neon Therapeutics. H.J.C. received commercial research support from Agenus Inc. and Genentech Roche and compensation for consulting/advisory boards from Genentech Roche, Celgene, Bristol-Myers Squibb, and GlaxoSmithKline. Other authors declare no financial disclosures. The authors declare no competing financial interests. Data and materials availability: N/A

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Table 1. Subject characteristics.

Age, median (range) 56 (33-66) Male (%) 69% Isotype, n (%) IgG 8 (62%) IgA 5 (38%) ISS stage (n=12), n (%) I , II, III 7 (58%), 3(25%), 2 (17%) Cytogenetics (n=11) *High-risk, n (%) 5 (45%) Standard-risk, n (%) 6 (55%) Induction regimen, n (%) VRD 4 (31%) VD or RD VRD 4 (31%) VDVCD 1 (8%) VRDVCD 1 (8%) VCD or VRDVCRD or VCTD 3 (23%) Response at enrollment, n (%) VGPR 12 (92%) CR 1 (8%) MAGE-A expression score, n (%) + (5-25% of CD138+ cells) 6 (46%) ++ (25-50%) 4 (31%) +++ (50-75%) 3 (23%) Stem cell mobilization regimen, n (%) G-CSF alone 1 (8%) G-CSF + plerixifor 9 (69%) Cyclophosphamide + G-CSF 3 (23%)

*Includes complex or hypodiploid karyotype, gain 1q, del17p, t(4;14). RD=lenalidomide/dexamethasone; VD=bortezomib/dexamethasone; VRD=bortezomib/ lenalidomide/dexamethasone; VCD = bortezomib/cyclophosphamide/dexamethasone; VCRD=bortezomib/cyclophosphamide/lenalidomide/dexamethasone; VCTD = bortezomib/ cyclophosphamide/thalidomide/dexamethasone

Figure Legends

Figure 1. Clinical outcome. Progression-free and overall survival (PFS and OS, respectively).

Median PFS was 27 months. Median OS not reached as of data lock.

Figure 2. Autologous lymphocyte infusion + MAGE-A3 vaccination elicits humoral

immune responses in autoSCT subjects. Peripheral plasma antibody titers by ELISA against

recombinant MAGE-A3 protein made in baculovirus (A) or overlapping pool of 20mer MAGE-

A3 peptides (B) show seroconversion in all subjects. Results were repeated independently at

least 3 times, with geometric mean and SEM shown. (C) Mapping of epitopes in indicated

subjects was performed by ELISA using individual overlapping 20mers, and reactive peptides

correlated with areas of hydrophilicity of the MAGE-A3 sequence. (D) Isotype and subclass of

immunoglobulin usage for MAGE-A3 antibodies showed IgG1 and IgG3 as the dominant

subclass, along with IgA and IgM detectable in some subjects either at d31 or d73, or at end of

study (EOS). (E) Titers against Protein D were present at baseline in some subjects but increased

significantly after treatment. Wilcoxon paired t tests indicate significant differences from

baseline (**: p<0.01, ***: p<0.001).

Figure 3. Autologous lymphocyte infusion + MAGE-A3 vaccine elicits Th1-biased CD4+ T-

cell immunity. Immune monitoring of peripheral CD4+ (A) T-cell responses to MAGE-A3 by

ELISPOT after in vitro sensitization (IVS), using autologous EBV-B cells pulsed with MAGE-

A3 OLP. Spots indicate average counts per 50,000 cells after subtraction of spots against EBV-B

target pulsed with irrelevant DMSO. (B) Statistical analyses of median and mean IFN+

ELISPOT numbers for CD4+ and CD8+ T-cell responses to MAGE-A3 or control NP after IVS

with respective cognate peptide pool, pooling all results from B-EBV and T-APC targets.

Wilcoxon paired t tests indicate significant differences from baseline (*: p<0.05, **: p<0.01,

***: p<0.001). Yellow circles: pre-autoSCT specimens (d-45 screening and d-24 leukopheresis.

Blue circles: post-autoSCT time points. (C) Ex vivo immune monitoring of T-cell responses from

PBMC show significant increase from baseline one year after treatment. (D) Assessing

polyfunctionality of CD4+ T cells against MAGE-A3 using cytokine secreted from supernatants

of ELISPOT cultures shown in panel A, after subtraction of values against DMSO target. Results

indicate significant induction of GM-CSF, TNFα, MIP-1β, IL5, and IL8 (Wilcoxon t test). (E)

Polyfunctionality of CD4+ T-cell responses to MAGE-A3 by intracellular staining for indicated

cytokines was confirmed by flow cytometry, as shown in example on top and by significant IFN

and TNFα presence (paired t test). (F) Quality of T cells responses to MAGE-A3 was assessed by

their capacity to recognize naturally processed MAGE-A3 full-length antigen either in the form

of recombinant viral vector (vaccinia or adenovirus) encoding MAGE-A3 after APC infection

(endogenous processing) or recombinant protein exogenously loaded to APCs. To characterize

CD4+ T-cell quality, MAGE-A3-specific cells were enriched from IVS cultures using CD154-

guided cell sorting. (G) MAGE-A3 specific CD4+ T-cell lines obtained after enrichment in three

subjects shown were able to recognize MAGE-A3 naturally processed protein, as well as protein

from viral vectors in S13 and S25.

Figure 4. Autologous lymphocyte infusion + MAGE-A3 vaccine does not induce robust

CD8+ T-cell immunity. (A) CD8+ T-cell responses to MAGE-A3 by ELISPOT after in vitro

sensitization (IVS), as in Fig. 2A. (B) CD8+ T cells from subject F06 at end of study show

capacity to recognize both viral constructs specifically for MAGE-A3, and not control NY-ESO-

1 antigen after EBV-B infection (Mann-Whitney t test). Bone marrow biopsy specimens from

selected subjects at screening (A and B) and relapse (C and D) were assessed for MAGE-A3 and

CD138 expression by IHC.

Figure 5. Co-stimulatory and co-inhibitory molecule expression changes in postSCT CD4+

populations. Peripheral blood mononuclear cells from depicted longitudinal milestones were

immunophenotyped by CyTOF. Cells were gated on CD4+ T-cell populations and PD-1.

CD45RA-/CCR7- Effector Memory (A) 4-1BB (B), ICOS (C), CD38 (D), PD-1 (E), and LAG3

(F) expression was quantified by percentage expression (left panels) and fold change from

screening (right panels). Wilcoxon paired rank test demonstrated significant increase of all

markers expression in these populations except LAG3. *: p<0.05, **: p<0.01, ***: p<0.001

1 0 0

8 0

6 0

4 0 PFS OS 2 0 Percent survival

0 0 10 20 30 40 50 M o n th s

Figure 1

Figure 2

Figure 3 Downloaded from cancerimmunolres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 11, 2019; DOI: 10.1158/2326-6066.CIR-18-0198 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4

** Wilcoxon paired ** Wilcoxon paired *** rank test rank test A. Effector Memory * *** ** D. CD38 * ** ** 80 F10 4 60 F10 F02 16 ** F02 F06 CD 4) F06 CD 4)

M08 cells 60 M08 8 on on M15 T 2 M15 M26 40 M26 4 CD45R A- M27 CD 4 M27 40 S02 S02 (CD38 2 S13 A- CD45R A- 1 on S13 S25 change Fold 20 e ng e 7- CCR 7- S25 on CD4 T ce lls on 20 S10 1 S10

% % S33 CD3 8

S33 cha

S36 (CCR7- -2

% % S36 0 -2 -45 -24 +31 +73 +194 +374 0

Day -45 -24 +31 +73 +194 +374 Fold Day -45 -24 +31 +73 +194 +374 -4 Injections pre 1 2 4 7 8 -45 -24 +31 +73 +194 +374 Injections pre 1 2 4 7 8 Days before and after auto-SCT (d0) Pre Post-transplant Days before and after auto-SCT (d0) Number of ASCI immunizations Number of ASCI immunizations Pre Post-transplant

*** Wilcoxon paired ns Wilcoxon paired rank test *** *** rank test B. 4-1BB * * ** E. PD-1 ** 12 F10 4 *** F10 8 ** F02 20 CD 4) F02

F06 15 CD 4) cells F06 M08 T cells 9 M08 on M15 10 4 2 T M15 M26 D-1 CD 4 M26 M27 8

(4-1BB on (4-1BB M27 (P 6 CD 4 S02 2

on S02 S13 6 S13 on e ng e S25 1 ng e S25

1B B 3 S10 4 S10 1 cha S33 cha S33 S36 2 d % 4- % % PD1 % S36 0 Fold -2 Fo l Day -45 -24 +31 +73 +194 +374 0 -2 -45 -24 +31 +73 +194 +374 Day -45 -24 +31 +73 +194 +374 -45 -24 +31 +73 +194 +374 Injections pre 1 2 4 7 8 Injections pre 1 2 4 7 8 Days before and after auto-SCT (d0) Days before and after auto-SCT (d0) Number of ASCI immunizations Pre Post-transplant Pre Post-transplant Number of ASCI immunizations

ns Wilcoxon paired ns Wilcoxon paired ns rank test *** rank test ns C. ICOS * F. LAG3 ns ** ns 15 F10 16 *** 5 F10 4 F02 F02 CD 4)

F06 CD 4) F06 cells cells 8 4 M08 M08 on

T 2 T 10 M15 M15 M26 4 M26 OS on OS 3 CD 4 CD 4 M27 M27 3 (LAG 3

(IC 1 S02 2 S02

S13 on 2 S13 e ng e 5 S25 ng e 1 S25 OS on S10 S10 -2

1 cha S33 cha -2 S33

% IC % S36 S36 LAG3 %

0 0 Fold -4 Day -45 -24 +31 +73 +194 +374 Fold -4 Day -45 -24 +31 +73 +194 +374 -45 -24 +31 +73 +194 +374 -45 -24 +31 +73 +194 +374 Injections pre 1 2 4 7 8 Injections pre 1 2 4 7 8 Days before and after auto-SCT (d0) Days before and after auto-SCT (d0) Pre Post-transplant Number of ASCI immunizations Pre Post-transplant Number of ASCI immunizations

Figure 5

Autologous lymphocyte infusion supports tumor antigen vaccine-induced immunity in autologous stem cell transplant for multiple myeloma

Adam D Cohen, Nikoletta Lendvai, Sarah Nataraj, et al.

Cancer Immunol Res Published OnlineFirst February 11, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-18-0198

Supplementary Access the most recent supplemental material at: Material http://cancerimmunolres.aacrjournals.org/content/suppl/2019/02/09/2326-6066.CIR-18-0198.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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