Abeome Corporation Page 1 of 17 Novel Humanized Monoclonal Antibodies to PD-L1 Using Abeome’s novel transgenic mouse antibody discovery platform, we have rapidly obtained antibodies of high affinity and neutralizing potency against human PD-L1 (Programmed Death Ligand 1, CD274). B-cells expressing affinity-matured anti-PD-L1 surface antibody were directly selected, and recombinant chimeric antibodies were immediately cloned and screened for PD-L1 binding and neutralization. With no additional optimization or affinity maturation steps, we generated humanized lead candidates that have comparable, and potentially superior, affinity and neutralization potency to atezolizumab, a humanized anti-PD-L1 mAb recently approved for the treatment of urothelial and non-small cell lung cancer (NSCLC). The further in vivo evaluation of these lead molecules should support a valid clinical development path. For licensing information, please contact us: Contacts Marty Simonetti [email protected]Kirby Alton [email protected]Rick Shimkets [email protected]Laboratories 111 Riverbend Road Athens, GA 30605 T- (706)542-7889
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Abeome Corporation Page 1 of 17
Novel Humanized Monoclonal Antibodies to PD-L1
Using Abeome’s novel transgenic mouse antibody discovery platform, we have rapidly
obtained antibodies of high affinity and neutralizing potency against human PD-L1 (Programmed
Death Ligand 1, CD274). B-cells expressing affinity-matured anti-PD-L1 surface antibody were
directly selected, and recombinant chimeric antibodies were immediately cloned and screened for
PD-L1 binding and neutralization. With no additional optimization or affinity maturation steps, we
generated humanized lead candidates that have comparable, and potentially superior, affinity and
neutralization potency to atezolizumab, a humanized anti-PD-L1 mAb recently approved for the
treatment of urothelial and non-small cell lung cancer (NSCLC). The further in vivo evaluation of
these lead molecules should support a valid clinical development path. For licensing information,
III. Immunization With Human PD-L1 Extracellular Domain IV. Harvest and Single Antigen-Positive Cell Sorting
V. High-Throughput Screening of PD-L1 Antibodies
VI. Identification and Characterization of Leads with PD-L1 Neutralizing Function
VII. Humanization Approach and Technology
VIII. Lead Characterization: Expression, Potency & Affinity
IX. PD-L1 Neutralization Primary T Cell Assay
X. Summary of Lead Discovery
XI. Therapeutic Product Development
I. PD-L1 (Programmed Death Ligand 1; B7-H1; CD274)
Research in the last two decades has established the concept that the PD-1/PD-L1
pathway acts to inhibit T cell activation and can be exploited by tumors to escape immune attack in
the tumor microenvironment [1]. Activation of PD-1 on the surface of T cells by engagement with
PD-L1 inhibits human T cell responses in vitro [2], and many in vivo studies have revealed that the
PD-1/PD-L1 pathway plays a major role in the suppression of T cell responses [3,4]. The PD-L1
protein is abundantly expressed on the cell surface in various human cancers, as indicated by
immunohistochemistry [5]. In contrast, normal human tissues seldom express PD-L1 protein on
their cell surface, with the exception of tonsil, placenta, and a small fraction of macrophage-like
cells in lung and liver [5,6]. Recent clinical success disrupting the PD-1/PD-L1 pathway has shown
the power of immunotherapy and brought about a sea-change in the treatment of cancer. The use
of combination immunotherapy may provide improved outcomes in patients over single-agent
checkpoint blockade. Such combinations may likely involve a PD-1/PD-L1 pathway blocker as its
backbone due to the remarkable therapeutic index of this class of agents [7].
II. Abeome Antibody Discovery Platform: AbeoMouseTM
We have developed a novel transgenic mouse system (AbeoMouseTM) allowing for the
direct selection of antigen-specific B-cells, paired with single-cell antibody gene cloning and screening. The AbeoMouseTM produces a 45-fold increase in surface immunoglobulin (Ig) positive antibody secreting cells and an accelerated immune response. Abeome’s screening platform allows 1,000 times more affinity matured monoclonal antibodies to be isolated from a single AbeoMouseTM than by conventional technology. In contrast to other current antibody technologies, this platform allows for the enrichment and rapid cloning of specific, high-affinity chimeric antibodies against a target of interest. With this modular system, cloned variable regions (V-regions) may be swapped between multiple human Ig isotypes for empirical comparison of stability, affinity and functional potency, or to suit the specific therapeutic modality or effector function.
Specifically, the transgenic AbeoMouseTM has been engineered to constitutively express multiple genes, including the Igα/Igβ B-cell receptor proteins, resulting in a hyper immune response and surface antibody expression during all stages of B cell differentiation (Fig.1). This enables the
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selection and sorting of antigen specific B-cells producing the most affinity matured antibodies, and this technology platform has been applied to obtain antibodies against a diverse set of antigens, including but not limited to whole cells, peptides, glycoproteins, viral envelope proteins and mouse proteins, typically producing chimeric leads with low picomolar dissociation constants.
FIGURE 1. The transgenic AbeoMouseTM platform. A novel antibody discovery platform that generates mature B cells with high surface IgG expression, allowing for the direct selection and cloning of antigen-specific B cells
III. Immunization With Human PD-L1 Extracellular Domain
Six AbeoMiceTM 7-8 weeks of age were pre-bled to obtain baseline serum antibody levels
and then immunized subcutaneously with 40 g of recombinant human PD-L1 extracellular domain (N-terminal segment Met 1-Thr 239 with a C-terminal polyhistidine tag; Sino Biological Inc.) in
proprietary adjuvant. Booster injections with 20 g of protein were given at days 14 and 28. Blood samples were taken and serum titers determined at days 21 and 35, resulting in extremely high titers, and mice were harvested at approximately 45 days after initial immunization (Fig. 2, Table 1).
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FIGURE 2. Immunization timeline for generating anti-PD-L1 antibodies
Table 1. Immunization and titer summary of mice immunized with PD-L1.
Male ms # 3341 10/09/2015 SC 10/23/2015 10/30/2015 >8,192K 11/6/2015 11/10/2015 > 8,192K
Male ms # 3342 10/09/2015 SC 10/23/2015 10/30/2015 >8,192K 11/6/2015 11/12/2015 > 8,192K
Male ms # 3352 10/09/2015 SC 10/23/2015 10/30/2015 >8,192K 11/6/2015 11/13/2015 8,192K
Male ms # 3353 10/09/2015 SC 10/23/2015 10/30/2015 8.192K 11/6/2015 11/13/2015 8.192K
IV. Harvest and Single Antigen-Positive Cell Sorting (Mouse #963)
As a representative example of this campaign, mouse #963 yielded 26 lymphoid tissue samples and bone marrow (Figure 3) which were harvested, processed, and pooled into a single suspension of lymphoid cells (6.6 x 108 cells). Erythrocytes were lysed using ammonium chloride and resulting lymphocytes were depleted of cells expressing IgM antibodies by immuno-magnetic separation.
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2 x 108 IgM depleted cells were labeled for FACS (fluorescent activated cell sorting).
Specifically, cells were first incubated with purified rat anti-mouse CD16/CD32 (mouse Fc
receptor block; BD Pharmingen) to block non-specific binding to mouse Fc receptors. Antigen
specific antibody was detected with ifluor-488 labeled recombinant human PD-L1. Surface
antibody was stained with alexa-fluor 647-GAMA-IgG. Antigen-positive cells were isolated as
single cells by FACS as illustrated in Fig. 4 (red boxed cell population). Cells expressing surface
antibody reactive to both human PD-L1 (ifluor-488) and GAMA-IgG (alexa-fluor 647) were singly
deposited into wells of a 96 well plate. A total of 1920 Ag+/Ig+ clones were sorted into wells from
this mouse.
FIGURE 4. Single cell FACS sorting of lymphocytes expressing antibody to human PD-L1 The direct selection of cells surface-expressing anti-PD-L1 antibodies is accomplished by staining harvested B cells with fluor-labeled recombinant human PD-L1 and a polyclonal antibody recognizing mouse surface immunoglobulin. Double-positive cells (red box) are sorted at 1 cell/well into 96 well plates for RT-PCR and cloning.
FIGURE 3. Lymphoid organs harvested from mouse #963 and processed into a single cell suspension for FACS (fluorescent activated cell sorting). Abeome’s transgenic mice have an enhanced immune response, greatly enlarged lymphoid organs (left), and typically an order of magnitude increase in the number of antibody producing plasmacytes.
Surface IgG
PD
-L1
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V. High-Throughput Screening of PD-L1 Antibodies
After cell sorting, twenty-seven 96-well plates of single B cells, comprising >2,000 antibodies, were subjected to nested RT-PCR using heavy and light chain variable region specific primer sets. Amplified V-regions were then fused with mammalian expression promoters and human Fc chains (IgG4 and kappa constant regions) by overlap PCR, generating transcriptionally-active PCR products. These individual paired heavy and light chain PCR products were transfected into HEK293 cells to generate supernatants of secreted chimeric antibodies and screened for binding to PD-L1 by ELISA. A representative set of screening data is shown (Fig. 5), which identified many antibodies positive for PD-L1 binding. In total, we isolated and cloned more than 60 chimeric monoclonal antibodies (mAbs) that showed strong binding to human PD-L1.
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FIGURE 5. Representative ELISA screening data for anti-PD-L1 chimeric antibodies. High-throughput screening of >1,000 novel antibodies revealed many with strong binding to human PD-L1. Values are expressed as human PD-L1 binding signal normalized to total IgG expression level.
VI. Identification and Characterization of Leads with PD-L1 Neutralizing Function
In order to identify potent, neutralizing antibody leads to human PD-L1, we utilized a PD-L1 neutralization reporter assay. Briefly, this two-cell system consists of a transgenic Jurkat line expressing a NFAT luciferase reporter and PD-1, and a CHO line expressing a TCR activator and human PD-L1 (Fig. 6). When co-cultured under normal conditions, the TCR activation signal in the Jurkat reporter line is blocked by engagement of PD-1 with PD-L1 on the cell surface, resulting in low luciferase activity. Neutralization of the PD-1/PD-L1 interaction by an antibody relieves this blockade and results in increased luciferase activity. The assay may be used to measure antibody potency by dose-dependent increase in luciferase activity.
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Figure 6. PD-L1 Neutralization Reporter Assay System
As a first step, the PD-L1 neutralization reporter assay was used to screen our set of novel PD-L1 antibodies at a single concentration (Figure 7) to identify all chimeric mAbs with neutralizing function. From this reporter assay screen, we identified 11 chimeric mAbs that resulted in strong activation, indicating neutralization of PD-L1. To further characterize these lead chimeric mAbs, we sequenced the mouse V-regions from our set of mAbs positive for PD-L1 binding. Interestingly, from the approximately 60 mAbs isolated from our screens, we classified eight sequence families, with a single sequence family representing all mAbs that exhibited PD-1/PD-L1 neutralizing activity in cell assay screens.
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Figure 7. Representative results from PD-L1 neutralization reporter assay screens. The figure shows the increased luciferase activity produced by 11 of the cloned antibodies. In the initial screen, approximately 60 antibodies exhibiting strong binding to PD-L1 were incubated with PD-L1 expressing-CHO TCR activator cells at a single concentration. This first cellular screen identified several candidate antibodies that showed neutralization of the PD-L1/PD-1 interaction. (add control description)
To identify the lead candidate chimeric mAbs with the highest potency in neutralizing PD-L1, we then performed dose titrations of the lead neutralizing mAbs in the reporter assay to determine IC50 values, and the activity of our lead mAbs was compared to a humanized clinical lead anti-PD-L1 mAb, atezolizumab. Atezolizumab (Tecentriq®) was developed by Genentech/Roche and was recently approved for the treatment of urothelial cancer and NSCLC. As shown in Figure 8, we identified a lead chimeric mAb, ABM101, with comparable activity to
atezolizumab. Specifically, ABM101 had an IC50 of 0.042 g/mL (290 pM) compared to 0.039
g/mL (269pM) for atezolizumab. In addition, we measured the affinity to human PD-L1 of ABM101 versus the clinical lead by surface plasmon resonance (SPR, Biacore). As we had shown for potency, we observed that ABM101 had a comparable affinity to atezolizumab in our experiments (Table 2).
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Figure 8. Neutralization of PD-L1 in a cell assay by lead chimeric mAb ABM101 is comparable to a clinical control, atezolizumab.
Table 2. Potency (IC50) and affinity (SPR) of lead chimeric mAb ABM101 compared to humanized clinical control antibody, atezolizumab.
VII. Humanization Approach and Technology
Abeome’s high-throughput monoclonal antibody discovery process begins with the cloning of mouse variable regions recovered from single B cells into proprietary human constant region-containing vectors, thus generating chimeric antibodies, which are tested for desirable properties. Ideally, multiple functional antibodies are selected to move forward into humanization, because in some cases it is not possible to design successful human framework grafts, while in other cases it is possible to obtain humanized antibodies with properties superior to the parent chimera.
Abeome’s approach to humanization comprises the generation of multiple heavy and light chain grafts, and pairwise testing to determine whether functional grafts can be immediately obtained. Structural models of each mouse fAb are generated and compared to the designed grafted fAb structure. As needed, back mutations are made in the grafted constructs in order to attempt to bring the structural models into alignment and maximize retention of parental affinity. Potential post-translational modification sites and other manufacturing challenges, such as non-canonical or unpaired cysteine residues and N-glycosylation sites are identified and engineered
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away. If additional affinity is required, mutagenesis of CDRs is undertaken using proprietary methods.
VIII. Humanized Lead Characterization: Expression, Potency & Affinity
Using our system of CDR grafting on human frameworks and proprietary expression vectors, we cloned a total of 6 heavy chain and 4 light chain humanized variations from the original ABM101 parent sequences. Many of the humanized antibody derivatives were subsequently cloned in IgG1 (N298A) and IgG4 (S228P) configurations. To characterize the humanized leads, we transiently expressed them in the Expi293 and ExpiCHO cell systems (Life Technologies) and measured the expression, potency and affinity of the purified mAbs. Examples of the potency and high affinity of the Abeome lead humanized candidates can be seen in Figures 9 and 10, respectively. Table 3 represents a summary of the potency and affinity data of the humanized leads. We did not see significant binding of any of the ABM101 family humanized mAbs to recombinant mouse PD-L1. In addition, we did not see any measurable binding of hABM101.11 to other human B7 family proteins (VISTA, B7-H3; data not shown).
Figure 9. Comparison of lead candidates A) hABM101.9 and B) hABM101.11 expressed as IgG1 and IgG4 isotypes to atezolizumab activity in the PD-L1 neutralization reporter cell assay.
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Figure 10. Multicycle Kinetics of ABM101.11 IgG1 and ABM101.9 IgG4 vs recombinant human (rhu) and cynomolgus (rcyno) monkey PD-L1. Affinity of anti-PD-L1 mAbs was
determined by surface plasmon resonance (SPR) on a Biacore T100. Anti-PD-L1 mAbs (25 g/mL) were captured onto a Protein A sensorchip and multicycle kinetics vs recombinant human or cynomolgus monkey PD-L1 (40 nM-2.5 nM) was determined. The antibody-PD-L1 complex was allowed to dissociate for 20 minutes before regenerating the surface with 10 mM glycine-HCL, pH 1.5. Data for ABM101.11 IgG1 and ABM101.9 IgG4 is shown.
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Table 3. Summary of humanized ABM101 family of anti-PD-L1 antibody chains, expression, potency (IC50), and affinity measurements. IC50 values are averages from multiple reporter cell assays, and affinity was measured by SPR binding to human and cynomolgus monkey recombinant PD-L1 extracellular domain. *KD values for atezolizumab were obtained from published reports [10, 11].
IX. PD-L1 Neutralization Primary T Cell Assay
To validate the function of our PD-L1 mAbs in primary human cells, we tested three of our
lead humanized mAbs in a primary human T cell assay. Studies by investigators in the field have
shown that PD-L1 extracellular domain coated on beads can inhibit the anti-CD3 proliferative
stimulation of and IFN secretion from T cells [8,9]. The neutralization function of antibodies may
be tested in this assay by blocking the negative effects of PD-L1. Briefly, magnetic beads were
coated with anti-CD3 antibody (OKT3), and then additionally coated with human IgG-Fc control
protein or human PD-L1-Fc protein. The coated beads were mixed with various concentrations of
purified anti-PD-L1 humanized mAbs, and the bead-antibody mixes were incubated with purified
human CD4+ T cells. After 5 days, cell proliferation (CellTiter Glo, Promega) and IFN secretion
(ELISA, R&D Systems) was measured, and antibody activity was assessed by neutralization of the
PD-L1 mediated decrease in cellular proliferation or IFN protein levels from control (IgG-Fc coated
beads). PD-L1 coated beads alone reduced proliferation by over 30% compared to the control IgG
beads, and all antibodies tested completely blocked the inhibition of proliferation by PD-L1 (Fig.
11A). Interestingly, the ABM101.11 and ABM101.9 mAbs show an increase in proliferation above
the control beads. For IFN secretion, we saw a modest decrease with PD-L1 beads, but this