Targeting B7-H3 in cancer

President and CEO
MacroGenics, Inc.
Rockville, MD

Targeting B7-H3 in cancer

by S. Koenig, USA

B7-H3 is a phylogenetically conserved protein with varied biological functions. In cancer biology, it appears to promote tumor cell invasion and metastasis and may modulate normal immune cell function. Expression of B7-H3 is pervasive in many solid tumors and its expression appears to be correlated with poorer clinical outcomes in some tumor types. Within both primary and metastatic tumors, B7-H3 may be expressed on multiple cell types, including the differentiated tumor cells, the tumor-initiating or cancer stem cells, and cells of the tumor vasculature. With promising clinical results using monoclonal antibodies directed to other members of the B7 family and their associated immune checkpoint coreceptors expressed on T cells, MacroGenics and Servier are pursuing the clinical development of an Fc-modified monoclonal antibody to B7-H3, called MGA271,which may impede tumor cell growth by various mechanisms. Phase 1 clinical studies are under way with the anticipated start of phase 2 development in 2015.

Medicographia. 2014;36:285-292 (see French abstract on page 292)

B7-H3 was first identified in 2001 from an analysis of a human expression database as a protein that was initially thought to function as a stimulator of T cells.1 Shortly thereafter, the naturally expressed form of human B7-H3 was revealed to encode a longer protein.2 Since the initial reports, B7-H3 has emerged as an important regulatory molecule for both normal and pathological conditions. The interest in B7-H3 is derived from its pleotropic effects in vertebrates spanning teleost fish to man with evidence of important biological functions in the emerging embryo through the aging adult.3 These include effects on fecundity and maturation of the embryo, skeletal system development,4 immune system function and modulation, and tumor cell invasion and metastasis. How a single protein could have such diverse functional roles is still an evolving question, but in large part is likely to be determined by mechanisms that regulate its expression, the sites in which it appears, and its putative receptors.

The B7 family and immune checkpoint inhibition

B7-H3 shares structural homology (approximately 20% to 30%) with other members of the B7 family, which are expressed to varying degrees on the cell surface of antigen-presenting cells of the immune system, but differ in their distribution on other cell types. A growing interest in B7-H3 has coincided with a large body of data published during the last few years in the rapidly developing field of immuno-oncology. Inhibition of immune checkpoints by monoclonal antibodies bound to inhibitory coreceptors expressed by T cells (eg, cytotoxic T-lymphocyte antigen 4 [CTLA-4] or programmed death-1 [PD-1]) or directed to their ligands (eg, PD-1 ligand [PD-L1]) within the B7 family resulted in powerful antitumor effects in several solid cancers in human clinical studies (Table I).5-8

Table I
Table I. Members of the B7 family of immune regulators.

Abbreviations: CTLA-4, cytotoxic T-lymphocyte antigen 4; ICOS, inducible costimulator;
NK, natural killer; PD-1, programmed death-1, VISTA, V-domain Ig
suppression of T-cell activation.

Significant numbers of patients with usually fatal tumors, such as melanoma, were apparently cured of their cancers.9 Recent data have demonstrated even more profound clinical effects when such therapeutics are combined.10 Thus, there is overwhelming interest not only to understand the best circumstances in which current clinical candidates directed to immune checkpoint molecules or their ligands can be exploited for therapeutic use, but to identify additional members of the B7 family and their receptors that could be targeted for clinical translational purposes.

B7-H3 and adaptive immunity

The initial focus on B7-H3 was prompted by its expression on cells of the immune system, particularly activated dendritic cells (DCs),11 and the manner in which it could modulate an endogenous adaptive immune response. Shortly after the report of the costimulatory properties of B7-H3 in enhancing T-cell proliferation, interferon (IFN)-ϒ induction, and cytotoxic T-lymphocyte responses, contradictory data began to emerge indicating that B7-H3 expression could inhibit T-cell–dependent responses, including studies performed in mice with knockouts of the B7-H3 gene.12 In particular, B7-H3–deficient mice developed more severe airway inflammation, earlier onset of experimental autoimmune allergic encephalomyelitis, and higher concentrations of anti-DNA autoantibodies compared with B7-H3–bearing animals.13 T-cell stimulation elicited with either anti-CD3 or allogeneic cells was inhibited by the presence of B7-H3, but this suppression could be compensated by costimulation of CD28 expressed on T cells. Similarly, in carefully conducted studies with human cells, B7-H3 suppressed T-cell proliferation and activation, especially in CD4+ T cells; however, attenuation of the inhibition was observed by the addition of interleukin 2 at the inception of the T-cell cultures.14 Beyond its effects on T cells, B7-H3 has been reported to inhibit natural killer (NK)-cell cytotoxic function, possibly by binding to an unidentified inhibitory receptor on NK cells.15

Various explanations could account for the observed disparity in the reported functional roles of B7-H3. It could be ascribed in part to structural differences within the molecule in humans compared with mice, the focus of most investigational functional studies. Mouse B7-H3 is a type 1 transmembrane protein containing 316 amino acids encoded by 2 immunoglobulin (Ig)-like exons; in humans, a duplication of the B7-H3 exons resulted in a larger 534-amino-acid molecule with 4 Ig-like domains (2 pairs of IgV-IgC), which in theory also could be alternatively spliced and expressed as a 2Ig-like protein, although evidence for significant expression of the latter is not well supported.16 Thus, the size, conformation, and avidity of human B7-H3 compared with the smaller murine version of this protein could account for differences in modulating immune responses. Indeed, in 1 report, cells constructed to express the 2Ig form of B7-H3 led to immune stimulation of human and mouse T cells, while other cells bearing 4Ig-B7-H3 molecules suppressed human T-cell responses.3 However, in a recent report describing the X-ray crystal structure of the murine B7-H3, inhibition of T-cell proliferation was observed with the protein used for crystallization and the functional inhibitory activity was mapped to a particular domain (ie, FG loop) of the B7-H3 molecule.17

B7-H3 is highly glycosylated with 4 predicted N-linked glycans increasing the size of the extracellular domain in the case of murine B7-H3 from 24 kDa to 40 kDa and in humans, up to about 100 kDa.17 It is possible that modifications in carbohydrate form or content among various tissue types, or with- in pathological tissues (such as tumors), or in different species could affect how B7-H3 engages other molecules and receptors. Most importantly, however, biological responses to B7- H3 could be significantly influenced by the particular receptors bound and engaged. Such receptors could program either activating or inhibitory responses and the outcomes could vary depending on receptor density and cell distribution. Although 1 putative receptor in mouse, called triggering receptor expressed on myeloid cell (TREM)-like transcript 2, has been reported,18 subsequent studies could not substantiate these findings either in mouse or human systems.14 In the absence of a confirmed receptor, it is therefore difficult to speculate on the absolute functional role for B7-H3 in normal and pathological conditions, especially when studied in isolation from other B7 family members and their coreceptors, which also participate in regulating human responses.

B7-H3 in cancer

In the context of cancer biology, a substantial literature has appeared over the past 5 years correlating the broad overexpression of B7-H3 on many solid cancers either with poorer clinical outcome or more advanced disease in these patients. These studies conducted by clinicians and pathologists include investigations of patients with prostate, ovarian, breast, colon, renal, non-small cell lung, pancreatic, and head and neck cancers, as well as melanoma, glioblastoma, and neuroblastoma and other small round blue cell tumors of childhood.19-31 In some of these studies, the poorer prognosis inversely correlated with immune infiltrates in the tumor, implying an untoward effect of B7-H3 expression with the generation of antitumor immune responses or migration or proliferation of inflammatory cells to the sites of tumors expressing B7-H3. Rarer studies (eg, those involving gastric and pancreatic cancer) have described improved outcome in association with B7-H3 expression. A notable observation is that for some solid tumors (eg, glioblastoma, renal cell, and ovarian carcinoma), B7-H3 is highly expressed within the tumor vasculature or in tumor-initiating cells of the central nervous system, and in these studies, patients were likely to have worse outcomes.32

Expression of B7-H3 in normal vs cancerous tissues

B7-H3 mRNA can be detected in various normal tissues, but the expression of B7-H3 protein in the healthy adult is very restricted. The disparity of ubiquitous detection of B7-H3 mRNA in normal and tumor tissues with the preferential expression of B7-H3 protein only in tumors may in large part be related to the levels of microRNA (miRNA)-29 in these tumors. An inverse relationship between miRNA-29 levels and B7-H3 protein was observed in normal and tumor tissues and cancer cell lines33; knock-in and knockdown experiments of miRNA-29 also led to down regulation and upregulation of B7-H3 protein, respectively, in tumor cell lines. In patients with melanoma, mRNA levels of B7-H3 were relatively increased in laterstaged disease and there was an associated inverse expression of miRNA-29c, suggesting that the reduced miRNA-29 may be largely responsible for promoting B7-H3 expression in cancer cells.34 Furthermore, the increased levels of B7-H3 in melanoma correlated with phosphorylated signal transducer and activator of transcription 3 (STAT3) activity. An additional study linking B7-H3 with the STAT3 signaling pathway indicated that chemoresistance to paclitaxel in breast cancer was associated with B7-H3 expression and phosphorylated STAT formation; further analysis also showed dependency on upstream signaling through phosphorylated Janus kinase 2 (Jak2) and the downstream expression of myeloid leukemia-1 (Mcl-1) and survivin, which affect apoptosis in the cancer cells.24 Similarly, increased sensitivity of a pancreatic cell line to gemcitabine was linked to reduced B7-H3 expression in vitro and in vivo and this was associated with higher levels of survivin, which promoted tumor cell death by apoptosis.35

B7-H3 as a targeted therapeutic in cancer

In selecting and exploiting a particular cancer-associated protein, like B7-H3, for the purpose of developing targeted therapeutics, different parameters should be considered. Some of these criteria may include whether or not a protein target of interest increases tumor growth, avoids triggering pathways which promote apoptosis or cell death, enhances tumor cell migration or metastasis, fosters new vessel formation, or evades immune surveillance mechanisms. In this context, B7- H3 may contribute to the formation of a cancer by: (i) altering a signaling pathway within cancer cells (and possibly within tumor-initiating stem cells), rendering them insensitive to intracellular molecules that promote cell death, partly through the Jak2/STAT3/survivin-dependent pathway; (ii) enhancing cancer cell migration and invasion of underlying tissues or stroma36,37; (iii) fostering neovascularization; (iv) promoting metastasis as a consequence of enhanced mobility and vessel formation; and (v) modulating the adaptive and innate immune responses during the evolution of the cancer from a primary lesion through its dissemination.

Given these varied properties, B7-H3 may be ideal for designing targeted therapeutics with a range of modalities. Furthermore, since B7-H3 expression appears to be limited in its distribution in normal tissues compared with tumors, this may contribute to a more favorable therapeutic window, with diminished potential for triggering of immune responses to normal tissues in conjunction with antibody-mediated blockade of B7-H3. Similarly, a cytotoxic therapeutic approach could also be contemplated as it can, in theory, more effectively avoid damage to the body’s normal physiology and function, a safety issue for many cancer treatments.

Which targeted therapeutic approaches might have promise in treating B7-H3–positive malignancies? Independent of its effects on antitumor immune responses, the majority of the published clinical literature indicates that reducing B7-H3 expression on the tumor cells and tumor vasculature should be beneficial. Current genetic methods using short-hairpin RNA (shRNA) and RNA interference (RNAi) to inhibit B7-H3, while useful tools in the laboratory, are currently impractical for targeting the widely distributed intracellular B7-H3 transcripts due to the inefficiency of these agents and the large copy numbers of inhibitors that would be required.

Figure 1
Figure 1. Tumor-specific reactivity of anti–B7-H3 monoclonal antibody.

Upper panel: Anti–B7-H3 murine monoclonal antibody (BRCA69D) demonstrates uniform and specific staining of freshly fixed paraffin-embedded prostate cancer specimen (left); absence of reactivity with BRCA69D when incubated along with soluble B7-H3 protein (middle) or with isotype control (right).
Lower panel: BRCA84D, the parental murine antibody of MGA271 shows intense staining of lung squamous carcinoma with the absence of staining in adjacent
normal lung tissue, as well as in normal spleen and lymph node tissues.
Abbreviation: mAb, monoclonal antibody.
Some of the data is derived from reference 42: Loo et al. Clin Cancer Res. 2012;18(14):3834-3845. © 2012, American Association for Cancer Research.

Another tactic could involve modulating B7-H3 molecules on the surface of cancer cells by an antibody, ligand, or even a small molecule, either by promoting the shedding of B7-H3, or through its reuptake and destruction within the tumor’s phagolysosomes. The loss of membrane-associated B7-H3 would abrogate any receptor-dependent signaling (of both the tumor cell itself and the putative coreceptors for B7-H3 expressed on immune cells). This latter approach assumes that there would be saturation of membrane-associated B7- H3 and its loss in expression would not be compensated by redistribution of molecules from the intracellular pool (often present in B7-H3–positive tumors) to the cell surface. It also presumes that only the membrane pool of B7-H3 promotes its tumorigenic effects, while any intracellular pool is inactive. A variation of this approach would be the blockade of stably expressed, membrane-bound B7-H3 molecules. Such an approach could mimic the therapeutic successes achieved with an antibody for PD-L1, the B7 ligand for PD-1, the inhibitory coreceptor on T cells.

Table II
Table II. Expression of B7-H3 in different types of cancer.

B7-H3 expression defined by evidence of specific staining with murine monoclonal
BRCA69D in ≥10% of tumor cells and/or ≥25% of associated vasculature,
with intensity criteria as follows: Neg (negative); 1+ (weak); 2+ (moderate);
3+ (strong). The B7-H3 positive rate = ≥1+ sample numbers/total tested sample
numbers x100%; 2+ or above positive rate = ≥2+ sample numbers/total
tested sample numbers x100%.
Some of the data is derived from reference 42: Loo et al. Clin Cancer Res.
2012;18(14):3834-3845. © 2012, American Association for Cancer Research.

Antibody-based treatments could be adopted for targeting B7-H3–expressing tumors by building on the recent clinical successes of coupling antibodies to cytotoxic agents for the treatment of lymphoma (ie, brentuximab vedotin) and breast cancer (ie, trastuzumab emtansine) or antibodies linked to radioisotopes, a method used for diagnostics as well as therapeutics for cancers. In fact, a cytotoxin conjugate has shown promise in treating glioblastoma, breast cancer, and osteosarcoma in small animal models, where a single- chain antibody to B7-H3 linked to a recombinant Pseudomonas immunotoxin had antitumor effects and survival benefit.38,39 Likewise, an 131I-labeled monoclonal antibody to B7-H3 was used to treat patients with intracerebral metastatic neuroblastoma with evidence of objective responses in some patients.40

Antibodies or antibody fragments that amplify their inherent immunological properties could be exploited for the killing of B7-H3–positive cancers. These could include complement mediated cytolysis and antibody-dependent cellular cytotoxicity (ADCC). In fact, a monoclonal antibody to CD20 (ie, obinutuzumab) with modifications of its Fc domain to enhance effector function was recently approved for the treatment of mantle cell lymphoma. Other more experimental approaches, such as the recruitment of cytotoxic T cells using bispecific technologies (eg, dual-affinity retargeting [DART] molecules)41 or genetically reprogrammed T cells using chimeric antibody receptors (CARs) could be adopted in this context.

In devising our lead therapeutic targeting B7-H3, we chose an initial approach that in principle could impede the suppressive effects on the adaptive (ie, T-cell mediated) immune response. In our characterization of about 50 monoclonal antibodies to B7-H3 generated by cell-based immunizations, we identified an antibody to a particular epitope that was found on most solid tumors, but had extremely limited expression on the 33 normal tissues examined by immunohistochemistry (Figure 1 and Table II).42

Given the selective tumor-binding specificity of this antibody, it gave us an opportunity to combine its potential to modulate an immune cell checkpoint with its potential to mediate antibody-dependent cellular cytotoxicity. Such cytotoxic activity could be directed toward the cancer cells, a pool of tumor- initiating or cancer stem-like cells, and any newly formed vessels within the tumor (Figure 2).

A chimeric version of this monoclonal with a human Fc domain was shown to mediate ADCC against tumor cell lines with human peripheral blood mononuclear cells from healthy donors as a source of effector cells. Subsequently, the variable domains of this monoclonal were humanized and we substituted 5 amino acids in non–surface-exposed positions within the Fc domain, which had been shown previously to enhance ADCC activity with other antibody specificities (eg, human epidermal growth factor receptor 2 [HER2]). This modified Fc domain substantially increases binding to an activating Fc receptor expressed by effector cells such as NK cells and macrophages (ie, CD16) and reduces binding to an inhibitory Fc receptor (ie, CD32B). This resulted in enhancement of ADCC activity in vitro and was particularly pronounced when effector cells were obtained from subjects with an allele of CD16 that shows diminished binding to and affinity for the native IgG1 Fc wild-type sequence (Figure 3, page 290). Moreover, the magnitude of ADCC activity was comparably effective against many different cancer cell lines, relatively independent of the density of B7-H3 expression, consistent with our previous reported experiences with other Fc-modified antibodies.

Figure 2
Figure 2. Mechanisms for inhibiting tumor growth by targeting B7-H3 with
Fc-optimized monoclonal antibody.

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CSC, cancer stem cell; MGA271,
Fc-optimized, humanized, monoclonal antibody to B7-H3; NK, natural killer.

To determine whether this enhanced ADCC function translated into improved in vivo activity, immunodeficient mice engineered to be deficient in murine CD16 and expressing a transgene of the human CD16 low-binding allele were engrafted with 7 different human tumor cell lines and then treated with varying concentrations of MGA271, the humanized, Fc-enhanced monoclonal antibody to B7-H3. MGA271 demonstrated potent inhibition of tumor growth, ranging from tumor regression to the slowing and stasis of tumor growth and this activity was shown to be both Fc-dependent and more profound than an antibody identical to MGA271, but without the Fc modifications.42

Figure 3
Figure 3. MGA271, an Fc-optimized monoclonal antibody, mediates enhanced ADCC activity against B7-H3–positive cancer lines.

Upper panel: FACS staining with anti–B7-H3 mAb of SK-MES-1 (lung), MDA-MB-468 (breast), and Raji (NHL) cancer cell lines. Lower panel: Percent cytotoxicity of cell lines indicated with MGA271 (Fc-optimized, humanized, monoclonal antibody to B7-H3), humanized B7-H3 monoclonal antibody with wild-type human IgG1 Fc, chimeric B7-H3 monoclonal antibody with wild-type human IgG1 Fc, or rituximab at the indicated concentrations in the presence of human PBMCs.
Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; E:T ratio, effect to target ratio; FACS, fluorescence-activated cell sorting; F/F, low-affinity 158F homozygous CD16A genotype; FITC, fluorescein isothiocyanate; NHL, non-Hodgkin lymphoma; LDH, lactate dehydrogenase; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells.
Some of the data is derived from reference 42: Loo et al. Clin Cancer Res. 2012;18(14):3834-3845. © 2012, American Association for Cancer Research.

Toxicology studies in cynomolgus monkeys treated either with a single administration or 4 once-weekly doses of MGA271 showed no significant adverse effects. Currently, a multicenter phase 1 dose-escalation clinical study in the United States is being conducted with MGA271 in patients who have failed standard therapies and whose tumors express B7-H3. In the first segment of the study, 26 patients with 15 different tumor types were treated with doses of MGA271 from 0.15 mg/kg to 15 mg/kg intravenously with up to 4 weekly doses during the initial cycle of therapy and no dose-limiting toxicity was observed. In the current phase 1B segment of the study, an increased number of patients (ie, 45) with particular tumor types are being enrolled and treated with 15 mg/kg of MGA271 weekly until progression of tumor is observed. It is anticipated that phase 2 development will begin in 2015 in multiple tumor types and in combination with other agents which may complement the biological activity of MGA271. In conclusion, there is strong scientific and clinical rationale for pursuing directed therapeutics to B7-H3. As in most successful treatments of cancers, a combination of strategies, including immune-based and cytotoxic agents directed to B7-H3– expressing tumors, may result in the most favorable outcomes for patients.

Acknowledgments: I would like to thank the team at MacroGenics for the development of MGA271 (including the enclosed figures) and Drs Ezio Bonvini, Paul Moore, and Jon Wigginton for reviewing the manuscript, and Ms Melinda Hanson for her assistance in preparing this submission.

Keywords: B7-H3; cancer; checkpoint inhibitors; immunotherapy; monoclonal antibody

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