Single-dose anti-PD-L1/IL-15 fusion protein KD033 generates ......2020/12/08 · 14, Bio Xcell), blocking antibodies, or combinations thereof. Anti-mouse CTLA-4 (clone 9D9, BioXcell)

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Single-dose anti-PD-L1/IL-15 fusion protein KD033 generates synergistic anti-

tumor immunity with robust tumor-immune gene signatures and memory

responses

Stella A. Martomo1*, Dan Lu

1, Zhanna Polonskaya

1, Xenia Luna

1, Zhikai Zhang

1, Sam Feldstein

1,

Radovan Lumban-Tobing1, Danielle K. Almstead

1, Faical Miyara

1, Jeegar Patel

1

1Kadmon Corporation, 450 East 29th Street, New York, NY 10016

*Corresponding author: [email protected]

Address: 450 East 29th

Street, New York, NY 10016

Running Title: Anti-PD-L1/IL-15 induces cytotoxic gene signature in tumors

Keywords: cytokine fusions, bi-functional antibodies, tumor-microenvironment, immuno-stimulatory

Conflict of Interest: All authors are current or former employees/interns of Kadmon and hold stock

and/or stock options or interests in Kadmon.

on July 1, 2021. © 2020 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 8, 2020; DOI: 10.1158/1535-7163.MCT-20-0457

mailto:[email protected]://mct.aacrjournals.org/

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Abstract

Immunocytokines hold great potential as anti-cancer agents as they utilize a specific anti-tumor

antibody to deliver an immune activating cytokine directly to the immunosuppressive tumor-

microenvironment (TME). We have developed a novel immunocytokine (KD033) comprised of a fully

human, high affinity anti-programmed death-ligand 1 (PD-L1) linked to the sushi domain of the human

IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033

surrogate (srKD033) and a non-targeting antibody (ntKD033) were also developed to investigate

mechanism of action in murine tumor models. Efficacy analyses showed a robust anti-tumor effect of

single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon

tumor model, single dose srKD033 produced durable anti-tumor immunity as evidenced by resistance

to subsequent tumor re-challenges. Mice responding to srKD033 treatment showed increased retention

of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15-induced expansion of cytotoxic

cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced

significant anti-tumor activity in murine carcinoma models that are non- or minimally responsive to

IL-15 or anti-PD-L1/PD-1 monotherapy.

Introduction

Programmed death-ligand-1 (PD-L1) and programmed death-1 (PD-1) interaction have been shown to

play non-redundant roles in reducing tumor-specific T-cell response in the TME (1-3). Blocking the

PD-1/PD-L1 interaction with anti-PD-L1 or anti-PD-1 agents increases cytotoxic T-cell activation

leading to long-lasting anti-tumor responses in some patients (4-6). Despite the demonstrated efficacy

of PD-1/PD-L1 immune checkpoint inhibitors (ICIs) in a variety of cancers, a large percentage of

patients do not respond or rapidly become refractory to these therapies (7). A rational strategy to



http://mct.aacrjournals.org/

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increase clinical responses and also reduce resistance to ICIs, is to combine ICIs with cytokines that

enhance T-cell activation while avoiding T-cell exhaustion/inactivation (anergy) (7,8). The selectivity

of some cytokines in expanding activated T cells make them ideal candidates for combination therapies

with ICIs (9).

Immunostimulatory cytokines such as IL-15 have demonstrated clinical activity in the treatment of

certain cancers (10,11). In patients with metastatic renal cell carcinoma (RCC), recombinant IL-15 (rIl-

15) produced a dramatic efflux of NK and memory CD8 T cells from circulating blood and produced

clearance of lung lesions in two patients without evidence of regulatory T cells (Treg) stimulation or

significant adverse events (12). IL-15 acts through its specific receptor, IL-15Rα, which is expressed

on antigen-presenting dendritic cells, monocytes and macrophages. The membrane-bound IL-15/IL-

15Rα complex is then trans-presented to neighboring NK or CD8 T cells that express the IL-15RβƔ

receptor to induce anti-tumor activity (13).

Unfortunately, the short half-life of rIL-15 and the limited availability of IL-15Rα in vivo have limited

its usefulness. Therefore, efforts to increase rIL-15 half-life, reduce systemic toxicity, and direct

activity to the TME are expanding. IL-15 shares two components of its receptor (the common gamma-

chain and the IL-2/15RβƔ complex) with IL-2 (14), which is effective in the treatment of RCC and

metastatic melanoma. Thus, IL-15 and IL-2 can mediate overlapping, differing or even opposing

functions, particularly in the early phases of T-cell activation when T cells enter their differentiation

program (15). Importantly, administration of rIL-2 requires careful management to limit severe

toxicities, such as cardiopulmonary toxicity and end-organ dysfunction (16). Unlike rIL-15, rIL-2 also

expands regulatory T cells (Tregs) which contributes to an immunosuppressive TME and prevents

generation of tumor immunity (10,11). It has been shown that complexation of the IL-15Rα sushi-




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domain with IL-15 increases IL-15 half-life and bioavailability in vivo (17) and is effective in

mimicking endogenous IL-15/IL-15Rα complex binding to IL2/15RβƔ-expressing cells (18).

In this report we describe biological activity and efficacy of a novel human anti-PD-L1/ IL-15Rα-IL-

15 immunocytokine (KD033) which binds to human and non-human primate (NHP) cells expressing

PD-L1. A KD033 surrogate (srKD033) which binds to human, NHP, and murine cells expressing PD-

L1 was also developed to evaluate treatment efficacy in syngeneic murine tumor models. KD033 and

srKD033 are engineered to minimize antibody-dependent cellular cytotoxicity (ADCC) and also

facilitate IL-15 trans-presentation from PD-L1-expressing cells. KD033 targeted anti-PD-L1 delivery

is expected to activate/expand cytotoxic T-cell memory as well as antigen presentation when PD-L1-

expressing immune and tumor cells are present at the tumor site. We expect this effect will lead to

robust anti-tumor and memory responses and reduced systemic toxicities.

Materials and Methods

IL-15 Fusion Molecules

Production of KD033, a high affinity anti-human/monkey-PD-L1; srKD033, a high affinity anti-

human/monkey/murine-PD-L1; and ntKD033, a non-targeting antibody is described in Supplementary

Methods 1. Anti-PD-L1 IgG1 sequences were ID 246, 250 and 267 (US Patent 10,407,502 B2). For

ntKD033, aa31-35 (ID267) were WYGMD instead of AYRMF. Validation of activity is included in

Supplementary Figure 1. All antibodies were of IgG1 class and were fused to the human IL-15/IL-

15Rα complex (Figure 1A). A LALA modification, in which leucine residues at positions 234 and 235

of human IgG1 Fc domain are substituted with alanine residues, was incorporated into the Fc region in

order to reduce FcƔ receptor binding thereby minimizing ADCC (19,20).




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Cell Proliferation Assay

Mouse splenocytes (Cellero) were used to measure IL-15-induced proliferation. Increasing

concentrations of fusion proteins were added to 50,000 thawed splenocytes/well in 96-well U-bottom

plates in IMDM, 10% FBS with 55 µM 2-Mercaptoethanol (Invitrogen). Proliferation was measured at

day four using CellTitreGlo (Promega). Data points represented average of duplicate wells and were

repeated three times.

Cell Lines for Murine in vivo Studies

Cell lines were routinely tested for mycoplasma before inoculation. Cell lines, sources, and culture

conditions used are described in Table 1 and Supplementary Methods 2.

In vivo Syngeneic Murine Tumor Studies

The maximal tolerated dose (MTD, antibody dose level that does not cause untoward toxicity or death)

of srKD033 and ntKD033 was determined using single escalating intravenous (i.v.) doses in naive or

CT26-bearing Balb/c mice. The MTD of srKD033 and ntKD033 was determined to be 3 mg/kg and 1

mg/kg, respectively. These doses, or lower, were used for all subsequent in vivo studies. The efficacy

of srKD033 was evaluated in eleven subcutaneously implanted syngeneic mouse models (n=10 per

arm). Tumor cells and mouse strains used are listed in the Supplementary Methods 2. Efficacy was

determined by comparing measured tumor volumes in srKD033-treated versus vehicle-treated mice.

Assessment of srKD033 efficacy in CT26-bearing Balb/c mice was repeated and compared to efficacy

observed with ntKD033, anti-mouse PD-L1 (developed by Kadmon), anti-mouse PD-1 (RMP1-14, Bio

Xcell), blocking antibodies, or combinations thereof. Anti-mouse CTLA-4 (clone 9D9, BioXcell) was

used with srKD033 in the 4T1 breast cancer model. All fusion proteins were administered as a single




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i.v. dose, unless otherwise noted. Anti-PD-L1 mouse anti-PD-1 and anti-CTLA-4 were administered by

intraperitoneal (i.p.) injection twice per week over a 3-week period.

To determine which immune cell populations contribute to srKD033 efficacy, antibodies that deplete

either CD8 T (YTS 169.4, Bio Xcell) or NK cells (Asialo GM1, ThermoFisher Scientific) were used as

described in Supplementary Figure 2A. Depletions were confirmed by flow cytometry of peripheral

blood as described in Supplementary Methods 2.

MOA studies were also performed using CT26 colon carcinoma bearing Balb/c mice. Mice were

treated with either a single i.v. dose of srKD033 or ntKD033, or two i.v. doses of anti-PD-L1 when

tumors reached approximately 130 mm3. At 7 days post-treatment, peripheral blood, spleens and

tumors from each group were collected for flow cytometry and immunohistochemistry (IHC). Details

of IHC methods and detection of srKD033/ntKD033 in serum are provided in Supplementary Methods

2.

All in vivo mouse studies were conducted for Kadmon by Crown Bioscience Inc. Animal care and use

protocols and any amendment(s) were reviewed and approved by the Institutional Animal Care and

Use Committee (IACUC) of Crown Biosciences. All animal studies were conducted in accordance

with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care

(AAALAC).

Flow Cytometry Analysis

Serially diluted KD033, srKD033, ntKD033 and, where indicated, the anti-PD-L1 antibody were added

to 50,000 cells/well. Binding was detected using Allophycocyanin (APC)-conjugated goat anti-human




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IgG (Jackson ImmunoResearch, 1:200 dilution) and evaluated using the Guava EasyCyte™ HT Flow

Cytometer with GuavaSoft 2.2.2. (EMD Millipore) for data analysis. Data points are the means ± SD

of duplicate measurements, and were repeated three times. Additional description for in vitro flow

cytometry analyses is provided in Supplementary Methods 1. Analysis of PD-L1 expression on tumor

cells and immune cell populations following treatment with srKD033 in vivo can be found in

Supplementary Methods 2. Antibodies used are listed in Table 2.

Immunohistochemistry

FFPE or OCT blocks of tumor samples were sectioned (4 µm thickness/section) with a manual rotary

microtome and processed using a Leica Bond RX autostainer. Antibodies used are listed in Table 2. All

stained sections were scanned with a NanoZoomer-HT 2.0 Image system (40x magnification;

Hamamatsu photonics). IHC images were analyzed using the HALOTM

Image Analysis platform.

Whole sections were analyzed and areas of necrosis were excluded. Total cell numbers (or area) and

IHC positive cells (or area) were scored. The IHC score represents the positive to total cell count/area

ratio in the examined section..

Gene Transcription Analysis

Tumors were isolated on day 7 after treatment, snapped frozen and used for RNA transcription using

the NanoString mouse PanCancer IO 360 immune-oncology panel. NanoString gene expression

analyses and tumor RNA extractions were conducted by Canopy Biosciences as described in

Supplementary Figure 3.

Statistics




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Group comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons test.

Comparison of tumor growth for treated and control groups were performed using RM ANOVA. The

log-rank (Mantel-Cox) test was used for survival calculations. Tests were done on GraphPad Prism

software (version 8.1.2). Significance is indicated as follows: ns (not significant), p

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murine tumor models with varying percentage of PD-L1 positive tumor cells (Figure 1E). srKD033

was not effective in EMT-6 breast or Renca kidney tumor models. However, anti-tumor activity as

measured by tumor growth inhibition (TGI) was observed in the other models (Figure 1F-L). In the

murine CT26 colon carcinoma model, single-dose srKD033 produced a curative effect as evidenced by

the absence of tumor mass in some mice (Figure 1F). A similar effect was observed in the H22 liver

tumor model (Figure 1H). These data show that srKD033 is efficacious in the treatment of tumor types

that express varied levels of PD-L1, consistent with anti-PD-L1 activity on tumor and/or immune cells

in TME (21,22). srKD033 also showed significant efficacy in the B16F10 and B16BL6 melanoma

models, which are known not to respond to ICIs (Figure 1J, K).

srKD033 is better tolerated than ntKD033

To compare the mode of anti-PD-L1/IL-15 (srKD033) action to IL-15 alone (ntKD033) and to

ntKD033-anti-PD-L1 combination, CT26 tumor-bearing mice were treated with single-dose srKD033

or ntKD033, repeat dose anti-PD-L1, or combination ntKD033+anti-PD-L1. Analysis of body weight

in mice treated with single-dose ntKD033 or srKD033 showed that ntKD033 was well tolerated up to 1

mg/kg; srKD033 was tolerated up to 3 mg/kg. Body weight loss observed in srKD033-treated mice (3

mg/kg) on Day 4 after treatment was largely recovered by Day 6 (Figure 2A). These findings are

consistent with data from srKD033-treated naïve Balb/c mice (Figure 1D). Notably, mice treated with

ntKD033 at 1 mg/kg showed a significant reduction in body weight at Day 4 that did not recover by

Day 6.

At the highest tolerated dose levels, srKD033 was more effective than ntKD033 in the CT26 colon

carcinoma tumor model (Figure 2B and C). Interestingly, to achieve an efficacy comparable to that

observed with single-dose srKD033 (3mg/kg), a combination of single-dose ntKD033 (1 mg/kg) and




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repeat dose anti-PD-L1 (10 mg/kg) was required (Figure 2D, E). However, mice treated with this

combination experienced significant increases in spleen weights, compared to srKD033-treated mice

(Figure 2G). In addition, at Day 7, human IL-15 serum levels were higher in ntKD033-treated mice,

compared to srKD033-treated mice, although this difference did not reach statistical significance

(Figure 2H). Both srKD033 and ntKD033 treatments resulted in comparable increases in CD8 T cells

in the spleen (Figure 2H). In contrast, levels of splenic NK cells were significantly higher in ntKD033-

treated mice (Figure 2I).

In a comparison of one srKD033 “best-responder” to one ntKD033 “best-responder” (indicated by a

“+” in the tumor volume plots, Figure 2, panels J and K, respectively), the srKD033 responder showed

an increase in CD8 T lymphocyte infiltration into and surrounding the tumor which was not observed

in the tumor from the ntKD033 responder (Figure 2J, K). Because the response associated with single

treatments of srKD033 or ntKD033 were highly variable, the extent of NK cell tumor infiltration

between srKD033 and ntKD033 responders did not reach statistical significance.

srKD033 increased both adaptive and innate immune cell activation in tumors

To further evaluate the srKD033 MOA, eight responders (%TGI>50) and five non-responders

(%TGI

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in srKD033 responders when compared to vehicle-treated mice. While an increase of CD8 T cells in

peripheral blood was observed in all treatment groups, the highest levels were observed in srKD033

responders. In contrast, the highest increase in NK cells was observed in both ntKD033 responder- and

non-responder mice. Immunohistochemistry showed CD8 T and NK cell infiltration into tumors of

both srKD033- and ntKD033-responders, but statistical significance was only achieved in the srKD033

responders (Figure 3C). Moreover, in srKD033-responders, more anti-PD-L1/IL-15 was retained and a

significant increase of B220+ cell tumor infiltration was observed (Figure 3C, D). Notably, changes in

the peripheral blood lymphocyte population of srKD033-treated mice was also observed in

cynomolgus monkeys treated with single dose KD033 by i.v. administration. In these studies, KD033

treatment produced dose-dependent increases in CD8 T, NK and NKT-like cells in the periphery at 7

days post-dose (Supplementary Table 1).

The NanoString gene expression platform was used to evaluate treatment effect on gene expression in

tumors. Overall changes in the gene expression profiles were relatively comparable between srKD033-

and ntKD033-treated mice (Supplementary Figure 3). Because these fusion proteins both contain the

IL-15/IL-15Rα complex, it is likely that the majority of the observed changes were mediated by the

activity of IL-15. A notable difference between srKD033- and ntKD033-treated mice was a

significantly higher increase in cytotoxic genes (Gzma, Gzmb, Gzme and Prf1) in both srKD033

responders and non-responders (Figure 3E). There was a higher expression of exhausted T-cell related

genes as well as of genes associated with innate immune response in srKD033-responders compared to

ntKD033-reponders (Figure 3F, Supplementary Figure 3). Expression of T-cell related genes was

significantly reduced in srKD033 non-responders compared to srKD033 responders (Figure 3F).




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To evaluate further the cytotoxic immune cell population critically involved in mediating efficacy of

srKD033, select immune cell populations in CT26 colon carcinoma bearing Balb/c mice were depleted

prior to srKD033 treatment. Efficacy following single-dose srKD033 treatment was then assessed in

survival analyses. Administration of a CD8 T cell depleting antibody decreased CD8 T cells, but also

increased NK cells in peripheral blood (Figure 3F). Antibody depletion of NK cells produced the

expected decrease of NK cells in the periphery without increasing the population of other immune cells

that were evaluated. Importantly, depletion of NK cells did not decrease the efficacy of srKD033 in the

CT26 colon carcinoma model as evidenced by unchanged survival. Meanwhile, depletion of CD8 T

cells produced a significant reduction in srKD033-mediated tumor growth inhibition and survival, but

not immediately or completely (Figure 3G), suggesting a complex involvement of immune cell

populations, in addition to CD8 T cells, in mediating srKD033 efficacy.

srKD033 produced durable memory responses

Both srKD033-responder and non-responder mice showed greater increases in CTLA-4 expression,

compared to ntKD033-treated mice (Figure 3E). The axis of PD-1/PD-L1/CD80/CTLA-4 interactions

in TME was recently described (23,24). The administration of anti-PD-L1 was shown to decrease

CD80 on antigen presenting cells (APCs) in the CT26 and 4T1 tumor models (24). As CTLA-4 gene

transcription was also increased in srKD033 non-responders, we sought to determine if administration

of anti-CTLA-4 in combination with srKD033 would produce an anti-tumor effect in the 4T1 model

where srKD033 monotherapy was ineffective. The regimen of srKD033 in combination with repeat

dose anti-CTLA-4 was indeed observed to significantly increase TGI in the 4T1 model (Figure 4A).

The combination of anti-PD-1 and anti-PD-L1 can be synergistic as they mediate overlapping and non-

overlapping interactions between tumors and immune cells (PD-1/PD-L1), or between immune cells in




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TME (PD-L1/CD80) (22). PD-1 expression is increased significantly in srKD033 responders (Figure

3F). To determine if continuous anti-PD-1 blocking would be synergistic with srKD033, the

combination of single or repeat doses of srKD033 with repeat dose anti-PD-1 was evaluated. The

combination of single-dose srKD033 (3 mg/kg) with repeat doses of anti-PD-1 showed synergistic

activity when compared to either treatment alone (Figure 4B). Repeat dosing of srKD033 (1 mg/kg)

with repeat doses of anti-PD-1 was effective to eliminate tumors in 30% of treated mice; whereas the

combination of single-dose srKD033 (3 mg/kg) with repeat dose of anti-PD-1 cured 60% of the mice

in this treatment group (Figure 4C, D). Mice demonstrating a curative effect following srKD033

monotherapy or srKD033/anti-PD-1 combination therapy were re-challenged with the same tumor

(CT26) without additional antibody administration. All mice rejected the CT26 re-challenge (Figure

4E).

To determine if a CT26-specific T-cell response was generated in the cured (tumor-free) mice, either a

CT26-specific gp70 tetramer or a non-specific β-galactosidase tetramer was used to stain CD8 T

splenocytes for flow cytometry (Figure 4F). A significant increase in gp70 tetramer CD8 T cells, but

not non-specific β-galactosidase CD8 T cells, was observed in mice treated with srKD033 or the

srKD033/anti-PD-1 combination, confirming the generation of a sustained CT26-specific CD8 T cell

memory response.

Discussion

In this report, we have evaluated biological activity and treatment efficacy of a novel PD-L1/ IL-15/IL-

15Rα immunocytokine (KD033). One of the features of this bi-functional antibody is its ability to

guide IL-15 to the TME, thereby stimulating intratumoral cytolytic immune responses. Data from our

studies has shown that treatment with the KD033 murine analog (srKD033) increases survival and




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tumor growth inhibition in multiple murine tumor models. In the TME, srKD033 enhances activation

of cytotoxic cells and induces a synergistic effect on innate and adaptive immune cell. By fusing IL-15

to anti-PD-L1, the effects of IL-15 in vivo shifted from changing the ratio of cytotoxic/Treg cells to

eliciting a robust activation of cytotoxic cells without significantly affecting Treg, resulting in the

generation of a potent memory response after single-dose srKD033 treatment.

KD033 and srKD033 differ from the recently described anti-PD-L1/IL-15 fusion protein, N-809 (25)

in that the mode of KD033/srKD033 action was not intended to involve ADCC or complement-

dependent cytotoxicity. In KD033/srKD033, IL-15 was linked to the C-terminal Fc-domain, rather than

the N-terminal. The C-terminal Fc-fusion likely preserves high affinity binding to PD-L1 (due to

opposite sites of binding) on either tumor or immune cells in the TME. The increased retention of

srKD033 in the TME is consistent with this MOA (Figure 3C). Accordingly, single-dose srKD033

treatment produced a varied level of efficacy in a variety of tumors, including tumors which are known

to be non-responsive to ICIs, such as the B16F10 melanoma model.

Previous studies have shown that IL-15 treatment, or combination treatment of IL-15 or IL-2 with PD-

1/PD-L1 blockade, increases expression of PD-L1 and produces robust anti-tumor responses in murine

cancer models (26,27). In the present study, single-dose srKD033 treatment in a CT26 colon carcinoma

model showed increased efficacy when compared to repeat doses of anti-PD-L1 alone, or the highest

tolerated dose of ntKD033. Durability of the srKD033 treatment response was superior to that

observed with ntKD033. Importantly, 3 mg/kg srKD033 was better tolerated than 1 mg/kg of

ntKD033, or combination ntKD033/anti-PD-L1, as assessed by changes in body and spleen weights.

These changes were coincident with significantly higher levels of IL-15 in serum (Figure 2G) and

increased percentage of splenic NK cells (Figure 2I), which was not observed following srKD033




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treatment, despite the lower dose of ntKD033 (1 mg/kg ntKD033 vs. 3 mg/kg srKD033). By targeting

IL-15 to PD-L1-expressing cells, greater localized immune stimulation was achieved with reduced

systemic toxicity (Figure 3E). In contrast, ntKD033 treatment resulted in prolonged presence of IL-15

in the periphery resulting in increased systemic NK cell stimulation in spleen and PBMCs (Figure 3B).

Depletion of CD8 T cells resulted in a significant increase of NK cells in the periphery following

srKD033 treatment (Figure 3F). This finding suggests that srKD033 changes the balance of cytotoxic

cell activation in the periphery. The ability of srKD033 to redirect IL-15 toward the TME differentiate

its mode of action from that of ICIs, free IL-15, modified free IL-2, or IL-15/IL-2 combination with

ICIs, which are currently being evaluated in various cancer indications.

Our data also suggest that srKD033 has a different mode of action in the TME compared to free rIL-

15, rIL-2 or ICIs. Recently it has been shown that anti-PD-L1 treatment disrupts the balance of PD-

L1/CD80 cis interaction (23,24) resulting in increased availability of B7.1 to bind to CTLA-4 on Tregs.

We hypothesize that when srKD033 is bound to PD-L1 expressing APCs in the TME, srKD033 can act

both as a PD-L1 blocker and a co-stimulatory molecule for CD8 T cells (Supplementary Figure 1H).

With co-stimulatory activity of srKD033 on APCs, even if PD-L1 blocking resulted in increased

CTLA-4/CD80 interaction (24), activation/proliferation of CD8 T cells could still occur. In the case of

srKD033 non-responders, srKD033 could be bound to PD-L1 expressing cells other than APCs. The

Tregs/CTLA-4 axis could play a significant role, resulting in less CD8 T cell activation/proliferation.

srKD033 could also directly increase activation of cytotoxic cells resulting in increased exhaustion. In

agreement with this hypothesis, co-administration of srKD033 with ICIs such as anti-CTLA-4 and anti-

PD-1 increased the anti-tumor efficacy of srKD033 (Figure 4A, B). In contrast to srKD033, the relative




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non-specificity of ntKD033 is consistent with the observed increase of cytotoxic cells in the periphery,

especially NK cells, which in turn non-specifically increase trafficking of these cells into tumors.

Data from our gene expression analysis revealed elevated levels of cytotoxic genes (Gzma, Gzmb,

Gzme and Prf1) in both srKD033-responsive and non-responsive mice, compared to ntKD033

responsive mice (Figure 3E). The absence of anti-tumor activity in srKD033 non-responders despite

the increased expression of cytotoxic genes suggests that increased levels of cytotoxic proteins in

tumors alone may not be sufficient to elicit an anti-tumor response. In addition to the efficient co-

stimulatory effect of srKD033 on CD8 T cells, the increased retention of anti-PD-L1/IL-15 in

srKD033-responsive tumors (Figure 3C) could facilitate prolonged and increased infiltration of other

immune cells such as B220+ cells in the TME (Figure 3C). The increase of B220+ cells observed in

the srKD033-responsive tumors correlated with the increase of cytotoxic genes identified in the gene

transcription analysis. This is consistent with these cells being pre-mNK cells possessing NK-like

killing with functional APCs capabilities (28,29). Furthermore, increased expression of genes such as

Itgax and Nos2 (Figure 3F) suggests involvement of both innate and adaptive immune response in

srKD033-responders.

The lower levels of NK cell proliferation in the periphery of srKD033-responsive mice, compared to

ntKD033-treated mice, did not decrease NK cell infiltration into tumors. The ability of some CD8 T

cells to acquire NK cell markers after exposure to IL-15 or IL-2 has been previously demonstrated

(30,31). It is possible that these cell types were induced by srKD033 treatment and could have

contributed to the observed efficacy. srKD033-responsive mice showed higher levels of NKT-like cells

in peripheral blood, compared to non-responsive and ntKD033-treated mice, and increased in NKT-

like cells was also seen in the periphery of KD033-treated cynomolgus monkeys (Supplementary Table




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1). The production of invariant NKT (iNKT) cells may account for this observation. The finding that

intratumoral iNKT-cell infiltration is a positive prognostic indicator in colorectal cancer (32,33) is

consistent with our findings in the CT26 colon carcinoma model and support the use of

immunophenotyping to predict and/or evaluate response to treatment. Additional studies are ongoing to

determine the exact nature of these NKT-like cell populations and their potential involvement in

KD033/srKD033-mediated tumor immunity.

Our PD-L1 targeting immunocytokine has been shown to increase T-cell activity via inhibition of PD-

1/PD-L1 interaction, deliver IL-15 to the TME leading to activation of T- and NK-cells locally,

achieve a robust and durable anti-tumor efficacy, and reduce toxicity associated with systemic IL-15

exposure. Additionally, clinical development of a single bi-functional, versus combination, therapy

would likely reduce cost and burden for patients and health care professionals. Because PD-L1 is

expressed in various cell types, the potential for off-target effects of KD033 in patients is of concern.

In this regard, it is important to note that repeat administration of srKD033 in mice, and repeat

administration of KD033 at therapeutic doses in monkeys, did not produce long-term adverse effects.

We believe that the biological activity, efficacy, and safety of KD033 support its continued

development toward clinical evaluation.

Acknowledgments

The study was supported by Kadmon. Editorial support was provided by Nathan Mata of Halloran

Consulting Group working on behalf of Kadmon.

References




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Table 1. Cell lines and mouse strains used for in vivo studies

Cell line Cancer type Cell source Media* Mouse

Strain Supplier




22

EMT-6 Breast ATCC (American Type Culture

Collection) DMEM BALB/C LC

CT26 Colon SIBS (Shanghai Institutes for Biological

Sciences) RPMI1640 BALB/C LC

H22 Liver CCTCC (China Center for Type Culture

Collection) RPMI1640 BALB/C LC

Pan02 Pancreatic NIH (National Institutes of Health) RPMI1640 C57BL/6 LC B16BL6 Melanoma Nanjing KeyGen Biotech Co., Ltd. RPMI1640 C57BL/6 LC

RM-1 Prostate SIBS (Shanghai Institutes for Biological

Sciences) RPMI1640 C57BL/6 LC

Renca Kidney ATCC (American Type Culture

Collection) DMEM BALB/C LC

B16F10 Melanoma SIBS (Shanghai Institutes for Biological

Sciences) DMEM C57BL/6 LC

MC38 Colon FDCC (FuDan IBS Cell Center ) DMEM C57BL/6 LC MBT2 Bladder RIKEN (RIkagaku KENkyusho) RPMI1640 C3H VR

LL/2 Lung SIBS (Shanghai Institutes for Biological

Sciences) DMEM C57BL/6 LC

4T1 Breast SIBS (Shanghai Institutes for Biological

Sciences) RPMI1640 BALB/C LC

* all with 10% FBS LC: Shanghai Lingchang Bio-Technology Co. Ltd (LC, Shanghai, China)

VR: Vital River Laboratory Animal Technology Co., Ltd. (VR, Beijing, China)

Table 2. Antibodies used for flow cytometry and immunohistochemistry

Markers Fluorochrome Clone Cat. Isotypes Vendor Note CD45 Percp-cy5.5 30-F11 103132 Rat IgG2b, κ Biolegend FC CD3 BUV395 17A2 740268 Rat IgG2b, κ BD FC CD4 FITC GK1.5 100406 Rat IgG2b, κ Biolegend FC CD8 APC-H7 53-6.7 560182 Rat IgG2a, κ BD FC FoxP3 PE FJK-16s 12-5773-82 Rat IgG2a, κ eBioscience FC CD25 APC PC61 102012 Rat IgG1, λ Biolegend FC CD335 BV421 29A1.4 137612 Rat IgG2a, κ Biolegend FC Live/Dead eFluor®506 NA 65-0866-14 N/A eBioscience FC Anti-PD-L1 BV711 M1H5 563369 Rat IgG2a, λ BD FC

gp70 tetramer APC H-2Ld

MuLV gp70 TB-M521-2 N/A MBL FC

β-gal tetramer PE H-2Ld β-gal TS-M511-1 N/A MBL FC

CD8 N/A 4SM15 14-0808-82 Rat IgG2a

mAb eBioscience

IHC,

1:400

CD335 N/A N/A AF2225 Goat pAb R&D IHC,

1:200

B220 N/A RA3-6B2 550286 Rat IgG2a

mAb BD

IHC,

1:500 Human-Fc N/A N/A ab98616 Goat pAb Abcam IHC, 1:50 ImmPRESS

HPR Anti-Rat N/A N/A MP-7444-15 N/A Vector IHC, 2ry




23

Ig ImmPRESS

HRP Anti-Goat

Ig N/A N/A MP-7405 N/A Vector IHC, 2ry

FC: Flow Cytometry IHC: Immunohistochemistry

Figure Legends

Figure 1. PD-L1 binding and IL-15 function of srKD033 in vitro and in vivo

A. Schematic representation of fusion proteins: KD033 and srKD033 (anti-human and -human/mouse

PD-L1 IgG1, respectively), and ntKD033 (an IgG1 antibody that does not bind to any known targets in

mouse), all fused to human IL-15/IL-15Rα. B. Analysis of fusion protein binding to PD-L1-expressing

mouse CT26 colon carcinoma cells. C. Human IL-15/IL-15Rα proliferation activity in murine

splenocytes. D. Analysis of body weight in Balb/c mice following a single i.v. dose of srKD033 (0.3, 1

or 3 mg/kg). E. Efficacy analysis in eleven different mouse models with varied tumor PD-L1

expression (measured when tumors reached 100 mm3) following a single i.v. dose srKD033 (3 mg/kg).

Tumor Growth Inhibition (TGI, %), was measured against vehicle treated animals on the day the first

animal in the control arm reached 3000 mm3 in tumor volume. F-L. Analysis of tumor growth

following a single i.v. dose srKD033 (3 mg/kg).

Figure 2. Tolerability and efficacy of single-dose srKD033 and ntKD033 in CT26 tumor-bearing

Balb/c mice

A. Body weight change in CT26 tumor-bearing Balb/c mice treated with single-dose ntKD033,

srKD033, anti-PD-L1, or combination treatment as indicated in the legend. B-E. Anti-tumor activity of

single-dose srKD033, ntKD033 (evaluated at their respective MTDs), or other treatments as indicated

in the legend. F. IL-15 levels in sera, and G. spleen weights in mice treated with single-dose srKD033,




24

ntKD033, or other treatments as indicated in the legend (see panel B). H, I. Measurement of splenic

CD8 T and NK cells in mice treated with single-dose srKD033, ntKD033, or other treatments as

indicated in the legend (see panel B). J, K. Tumor volumes and corresponding IHC analysis (panels to

right of E and F) of tumors from CT26 Balb/c mice treated with srKD033 or ntKD033.

Figure 3. Analysis of tumor histochemistry, peripheral immune cell markers and gene expression

in mice treated with srKD033 and ntKD033

A. Tumor growth of mice designated responders and non-responders after srKD033 or ntKD033

treatment. B. Flow cytometry of peripheral blood cells from srKD033 and ntKD033 responders and

non-responders. C, D. IHC analysis of CD8, NK, and B220+ cells, and fusion protein levels in tumors

from CT26 tumor-bearing Balb/c mice treated with single-dose srKD033 or ntKD033. E. Tumor

growth and NanoString analysis of gene expression changes in CT26 tumors. F. Volcano plots

showing changes in gene expression between srKD033 and ntKD033 responders (top) and srKD033

responders and non-responders (bottom). G. Flow cytometry of peripheral blood after CD8 T or NK

cell depletion prior to srKD033 treatment in CT26 model. G. Effect of CD8 T and NK cell depletion

on srKD033-mediated efficacy and survival in the CT26 model.

Figure 4. Efficacy and durability of srKD033 treatment in the murine colon carcinoma model

A. Analysis of tumor growth in orthotopically-implanted 4T1 tumor-bearing Balb/c mice treated with

srKD033 and anti-CTLA-4 combination. Analysis of tumor growth (panel B, spider plots in panel D)

and survival (panel C) in CT26 tumor-bearing Balb/c mice treated with different treatment regimens of

srKD033 or anti-PD-1. D. Analysis of tumor growth in CT26 mice cured by treatment (tumor-free

mice) following re-challenge with CT26 (1st), and H22 (2

nd) tumors. E. Measurement of Gp70+ CD8 T




25

cells specific for CT26, compared to levels of non-specific B-galactosidase+ CD8 T cells in

splenocytes of mice treated with either srKD033 or srKD033+anti-PD-1.




Published OnlineFirst December 8, 2020.Mol Cancer Ther Stella A Martomo, Dan Lu, Zhanna Polonskaya, et al. gene signatures and memory responsessynergistic anti-tumor immunity with robust tumor-immune Single-dose anti-PD-L1/IL-15 fusion protein KD033 generates

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Single-dose anti-PD-L1/IL-15 fusion protein KD033 generates ......2020/12/08 · 14, Bio Xcell), blocking antibodies, or combinations thereof. Anti-mouse CTLA-4 (clone 9D9, BioXcell)