Conditionally active antigen binding polypeptides to a b7-h3 (CD276) antigen, compositions, and methods of use thereof

Abstract

A pH-dependent conditionally active antigen binding polypeptide to a B7-H3 (CD276) antigen having a heavy chain variable region and / or light chain variable as well as antigen binding antibodies and antigen binding antibody fragments thereof containing the heavy chain variable region and / or the light chain variable region that bind to B7-H3 protein and multi-specific antibodies and antigen-binding antibody fragments thereof that bind to B7-H3 protein and bind CD3 optionally also in a pH-dependent conditionally active manner. Pharmaceutical compositions, methods of treatment and kits comprising the polypeptides, antibodies and antigen-binding antibody fragments thereof, multi-specific antibodies, and antigen-binding fragments thereof are also provided.

Description

BIAT-1039WO PATENT APPLICATIONCONDITIONALLY ACTIVE ANTIGEN BINDING POLYPEPTIDES TO A B7-H3 (CD276)ANTIGEN, COMPOSITIONS, AND METHODS OF USE THEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional application no. 63 / 755,145. filed on February 6, 2025, the disclosure of which is hereby incorporated by reference in its entirety as if fully set forth herein.INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing in ST.26 XML format entitled BIAT1039USP.xml, created on January' 17, 2025, comprising 25,465 bytes, prepared according to 37 CFR 1.822 to 1.824, submitted concurrently with the filing of this application, is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to anti-B7-H3 (CD276) polypeptides, anti- B7-H3 (CD276) antibodies and antigen-binding antibody fragments thereof, anti- B7-H3 (CD276) multi-specific antibodies, and antigen-binding antibody fragments each of which exhibit pH-dependent conditional binding activity to the B7-H3 antigen, and uses of these polypeptides, antibodies, antigen-binding antibody fragments, multi-specific antibodies and antigen-binding fragments thereof in pharmaceutical compositions and therapeutic methods.BACKGROUND

[0004] B7-H3 (B7 Homolog 3 Protein, CD276) is a type 1 transmembrane glycoprotein with two isoforms comprised of an extracellular V- and C-like Ig domain and is a member of the B7 family of immune checkpoint (IC) proteins that exert pleiotropic immunomodulatory effects both in physiologic and pathologic contexts. The protein encoded by this gene belongs to the immunoglobulin superfamily and is thought to participate in the regulation of a T-cell-mediated immune response. The B7-H3 protein is expressed in resting fibroblasts, endothelial cells, osteoblasts, amniotic fluid stem cells and other non-immune cells, and on the surface of induced antigen-presenting cells and NK cells. Many studies have revealed that B7-H3 is overexpressed in a variety' of tumors, including melanoma, pancreatic cancer, breast cancer, prostate cancer, colorectal cancer, and its expression level is closely related to a poor prognosis and the clinical outcome of patients. Preclinical studies have confirmed that the expression of CD276 mRNA in pancreatic cancer tissue is significantly higher than that of normal adjacent groups. Studies show that while theBIAT-1039WO PATENT APPLICATIONtranscript of this gene is widely expressed in normal tissues and solid tumors, the protein is preferentially expressed in tumor tissue.

[0005] Expression of B7-H3 in various solid malignancies, including tumors that have proven to be less sensitive to current immunotherapeutic options, has led to association of B7-H3 expression with advanced disease, lower patient survival rates and impaired response to immune checkpoint inhibitor (ICI)-based regimens. Anti-B7-H3 agents, including novel monoclonal antibodies (mAbs), bispecific antibodies, ADCs, CAR-T cells, and radioimmunotherapy agents, have exhibited encouraging antitumor activity in preclinical models and have recently entered clinical testing for several cancer types. See Feustel et al., “B7-H3 Inhibitors in Oncology Clinical Trials: A Review,” Journal of Immunotherapy and Precision Oncology' 7(l):53-66 (2024) (‘‘Feustel et al.”) and Koumprentziotis et al., “New Emerging Targets in Cancer Immunotherapy: The Role of B7-H3,” Vaccines 12:54 (2024) (“Koumprentziotis et al.”). Table 1 provides an overview of the state of development of anti-B7-H3 antibodies for the treatment of a variety of cancers, particularly various sarcomas. To date, there are no clinically available therapeutics / drugs targeting B7-H3 in any solid tumors.Table 1: Drugs that target B7-H3 undergoing clinical trials (adapted from Feustel et al. and Koumprentziotis et al.)BIAT-1039WO PATENT APPLICATION

[0006] International Patent Publication No. WO 2019 / 241216 (WO ‘216) describes multi-specific antibodies and methods of preparation for Axl, EpCAM, Ror2, Her2, and B7-H3, antigens in addition to more than eighty other cancer cell specific antigens. The publication further provides a multispecific antibody with conditional binding activity to CD3 and anon-conditional B7-H3 binding activity. WO '216 does not provide a pH-dependent conditionally active anti-B7-H3 polypeptide as claimed nor are the anti-B7-H3 polypeptides homologous to those of the present application.

[0007] Antibodies have become a major class of therapeutic proteins and as presented above, a number of antibodies targeting B7-H3 for use in cancer therapies have been developed and are undergoing testing. Traditional antibodies normally bind to a single epitope on an antigen. Multispecific antibodies have also been developed for binding to more than one antigen or to more than one epitope on the same antigen. Multi-specific antibodies may be, for example, bispecific, trispecific, or tetra-specific antibodies. Multi-specific antibodies have shown potential in abroad range of clinical and diagnostic applications. Due to their unique features, multi-specific antibodies have become attractive for next generation antibody therapeutics.

[0008] Obrindatamab (MGD009 or orlotamab) is a bi-specific mAb-like agent that simultaneously targets the B7 immune regulator B7-H3 (CD276) and CD3e. CD3e is expressed by immune effector cells and is a negative regulator of T-cell activation with a role in immune-evasion, tumor cell invasion and metastasis. MGD0009 was developed for its anti-tumor potential. See Malapelle, et al. , ‘'B7-H3 / CD276 Inhibitors: Is There Room for the Treatment of Metastatic Non-Small Cell Lung Cancer?” Int. J. Mol. Sci. 23(24): 16077 (2022). The proposed mechanism of obrindatamab is toBIAT-1039WO PATENT APPLICATIONpromote the activation and cell-killing functions of immune effector cells and direct this towards the co-engaged tumor cells. The peptide sequences for the heavy and light chains of the B7-H3 binding domains of obrindatamab are disclosed in U.S. Patent No. 8,802,091, issued August 12, 2014, to Johnson et al., and assigned to Macrogenics, Inc, as SEQ IDs 99 and 89 respectively and corresponding to the antibody BRCA84D).

[0009] US Patent Publication No. 2013 / 0017200 discloses a method of synthesizing multi-specific antibodies. A first antibody fragment is obtained from a first parent antibody having a first monospecificity, which has a free sulfhydryl group that may be reacted with a thio-reactive crosslinker to produce an antibody fragment-crosslinker moiety. The antibody fragment-crosslinker moiety is reacted pairwise with each of two or more additional antibody fragments obtained from other parent antibodies having a mono-specificity that is different from the first antibody fragment, each having a free sulfhydryl group, to produce the multi-specific antibodies. These multi-specific antibodies may be suitable as new therapeutic and diagnostic agents.

[0010] Brinkmann and Kontermann (“The making of bispecific antibodies,” MABS, 2017, vol. 9, pp.182-212, 2017) surveys formats of bispecific antibodies, including small molecules composed solely of the antigen binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. Depending on different applications, the bispecific antibodies may vary' in size, arrangement, valence, flexibility and geometry' of their binding modules, as well as in their distribution and pharmacokinetic properties.

[0011] It is desirable to generate useful antibodies that are conditionally active. For example, antibodies virtually inactive at a normal physiological condition and significantly more active at a condition other than the normal physiological condition or are activated or inactivated in certain microenvironments (e.g, activated in a tumor microenvironment), or antibodies that are activated or inactivated over time. Besides temperature, other trigger conditions for which the conditionally active antibodies are evolved or optimized include pH, osmotic pressure, osmolality, oxidative stress, oxygen concentration and electrolyte concentration. Besides the activity, other desirable properties of antibodies that can be optimized during evolution include expression level, stability, half-life, chemical resistance, and proteolytic resistance.

[0012] Many strategies for evolving or engineering a parent antibody to mutant antibodies having a desired property' have been published. However, engineering or evolving a parent antibody to be inactive or virtually inactive (e.g. less than 10% activity or especially less than 5% activity) at the normal physiological condition, while having activity at an aberrant condition that is equivalent or better than the original activity of the parent antibody at the normal physiological condition, requiresBIAT-1039WO PATENT APPLICATIONthat destabilizing mutation(s) co-exist with activity increasing mutations that do not counter the destabilizing effect. It is expected that destabilizing mutations would reduce the antibody’s activity by an amount greater than is predicted by standard rules such as the Q10 rule. Therefore, the ability to evolve proteins that work efficiently (are more active) at a specific aberrant condition, e.g. a lower temperature or pH, than the evolved protein at a normal physiological condition, or even being substantially inactive at the normal physiological condition, creates a surprising new class of proteins referred to as conditionally active proteins. These conditionally active proteins may have a ratio of activity at the aberrant condition to the activity at the normal physiological condition greater than the same ratio for the parent protein from which they are evolved.

[0013] The present application provides a new class of a pH-dependent conditionally active antigen binding polypeptides having binding activity’ the B7-H3 (CD276) antigen. Also included are pH-dependent conditionally active multi-specific antibodies that exhibit pH-dependent conditional binding to B7-H3 and, optionally, also to the CD3 antigen. This new class of multi-specific antibodies takes advantage of the flexibility’ and versatility’ of traditional multi-specific antibodies, while at the same time directing the binding activity, affinity’ and / or avidity of the multi-specific antibodies to locations, tissues or organs of a subject where the activity is needed.SUMMARY OF THE DISCLOSURE

[0014] The present application produces and includes an isolated polypeptide that specifically binds to B7-H3 and has a pH-dependent conditionally active B7-H3 (CD276) antigen binding activity. The isolated polypeptide comprises a heavy chain variable region comprising three complementarity' determining regions (CDRs) having amino acid sequences Hl, H2, and H3, whereinthe Hl sequence is GFDFSSFGEH (SEQ ID NOT);the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NOT); andthe H3 sequence is GRENIYYGARLDY (SEQ ID NOT); anda light chain variable region including three complementarity' determining regions having amino acid sequences LI, L2, and L3, whereinthe LI sequence is KASQNVDDQVA (SEQ ID NO:4);the L2 sequence is SASYRYS (SEQ ID NO:5); andthe L3 sequence is QQYNKYPFT (SEQ ID NO:6); and wherein the isolated polypeptide has a higher binding activity' to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0015] In an aspect, the present application includes isolated polypeptides wherein the pH-dependent conditionally active antigen binding activity’ to B7-H3 (CD276) comprises pH-dependent conditional binding activity to both human and cynomolgus monkey B7-H3 (CD276).BIAT-1039WO PATENT APPLICATION

[0016] In aspects, the pH-dependent conditionally active B7-H3 antigen binding polypeptides comprise a light chain variable region that includes the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:17.

[0017] Also included in the present application are pH-dependent conditionally active B7-H3 antigen binding polypeptides having a heavy chain variable region that includes the CDRs having the sequences Hl, H2 and H3 and has an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 15, and SEQ ID NO: 18.

[0018] In another aspect, the pH-dependent conditionally active B7-H3 antigen binding polypeptides comprise a light chain variable region having an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NO: 17.

[0019] In further aspects, the pH-dependent conditionally active B7-H3 antigen binding polypeptides include a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 18.

[0020] Also described are pH-dependent conditionally active B7-H3 antigen binding polypeptides that include variations of the amino acid sequences SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO:15, and SEQ ID NO:18, created by making conservative amino acid substitutions outside of the CDRs of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 18.

[0021] The present application includes, pH-dependent conditionally active B7-H3 antigen binding polypeptides that are antibodies or antigen binding fragments thereof including a heavy chain variable region having three complementarity determining regions (CDRs) with amino acid sequences Hl, H2, and H3, wherein the Hl sequence is GFDFSSFGEH (SEQ ID NO: 1); the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NO:2); and the H3 sequence is GRENIYYGARLDY (SEQ ID NO:3); and a light chain variable region including three complementarity determining regions having amino acid sequences LI, L2, and L3, wherein the LI sequence is KASQNVDDQVA (SEQ ID NO:4); the L2 sequence is SASYRYS (SEQ ID NO:5); and the L3 sequence is QQYNKYPFT (SEQ ID NO:6); and wherein the antibodies or antigen binding fragments thereof have a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0022] In an aspect, the present application includes antibodies or antigen binding fragments wherein the pH-dependent conditionally active antigen binding activity to B7-H3 (CD276) includes pH-dependent conditional binding activity to both human and cynomolgus monkey B7-H3 (CD276).BIAT-1039WO PATENT APPLICATION

[0023] In aspects, the antibodies or antigen binding fragments thereof having pH-dependent conditionally active B7-H3 antigen binding activity’ is a chimeric antibody, a multispecific antibody, or a humanized antibody, or fragments of any of those. In aspects, the multispecific antibody is a bispecific antibody or antigen binding fragment thereof.

[0024] In further aspects, the pH-dependent conditionally active B7-H3 antigen binding polypeptides are multi-specific and include binding activity to the CD3 antigen. In one embodiment, the binding activity to the CD3 antigen is pH-dependent conditional binding activity. In this embodiment, the peptide further includes a light chain variable region including six anti-CD3 complementarity determining regions having sequences L4, L5, L6, L7, L8, and L9, wherein the L4 sequence is GFTFNTYAMN (SEQ ID NOT), the L5 sequence is RIRSKYNNYATYYADSVKD (SEQ ID NO: 8), the L6 sequence is HSNFGNSKVSWFAY (SEQ ID NO:9) or HTNFGNSKVSWFAY (SEQ ID NOTO), the L7 sequence is RSSAGAVTTSNYDN (SEQ ID NO: 11), the L8 sequence is GTNKRAP (SEQ ID NO: 12), and the L9 sequence is ALWYSNLWV (SEQ ID NO: 13). The light chain variable region including the six anti-CD3 complementarity’ determining regions having the sequences L4, L5, L6. L7. L8. and L9 binds to CD3 antigen and the isolated polypeptide has a higher binding activity to the CD3 antigen at pH 6.0 than at pH 7.4.

[0025] In some aspects, the light chain variable region including the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8 and L9 is selected from a single chain variable fragment (scFv), a nanobody (Camelid VHH), a DARPIN, a minibody, or a tri-scFv, each having the pH-dependent conditional binding activity to CD3 antigen.

[0026] In some other aspects, the pH-dependent conditionally active B7-H3 antigen binding polypeptides include a light chain variable region having the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8 and L9 that is an scFv. The light chain variable region having the six anti-CD3 complementarity’ determining regions having the sequences L4, L5, L6, L7, L8 and L9 may be at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical, to an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO:20. In this embodiment, the CDRs are unchanged.

[0027] In another aspect, the polypeptides having pH-dependent conditionally active B7-H3 antigen binding activity and pH-dependent conditionally active anti-CD3 antigen binding activity are bispecific antibodies or antigen binding antibody fragments thereof.

[0028] In yet another aspect, the present disclosure provides a pharmaceutical composition that includes any of the isolated polypeptides, the antibodies or antigen binding antibody fragments of the application described above, together with a pharmaceutically acceptable carrier.BIAT-1039WO PATENT APPLICATION

[0029] In some aspects, the pharmaceutical composition of the preceding paragraph is a liquid form, a lyophilized form, or a liquid form reconstituted from a lyophilized form.

[0030] In some aspects, the pharmaceutical composition further includes an immune checkpoint inhibitor. The immune checkpoint inhibitor may be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, 0X40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H4, KIR, A2aR, CD27, CD70, DR3, or ICOS.

[0031] In an aspect, the present application provides methods of treating cancer comprising administering the isolated polypeptides, antibodies, antigen binding antibody fragments, multispecific antibodies or pharmaceutical compositions described above to a patient in need of treatment for cancer. The cancer is selected from carcinoma, adenocarcinoma, glioma, lymphoma, melanoma, or cystadenoma.

[0032] In another embodiment the cancers are selected from Carcinoma (CA), Castration-Resistant Prostate Cancer (CRPC), Central Nervous System (CNS) / Leptomeningeal Metastases (LM), Clear Cell Carcinoma (CCC) of the ovary, Colorectal Cancer (CRC), Epithelial Ovarian Cancer (EOC), Esophageal Squamous Cell Carcinoma (ESCC). Head And Neck Squamous Cell Carcinoma (HNSCC), Head And Neck Squamous Cell Carcinoma (HNSCC). Lentigo malignant Melanoma (LMM), Medulloblastoma (MB), Melanoma, Metastatic Castration-Resistant Prostate Cancer (mCRPC), Muscle Invasive Bladder Cancer (MIBC), Neuroblastoma (NBL), Non-Muscle Invasive Bladder Cancer (NMIBC), Non-Small Cell Lung Cancer (NSCLC), Prostate Cancer (PCa), Renal Cell Carcinoma (RCC). Retinoblastoma (Rb), Rhabdomyosarcoma (RMS), Small Cell Lung Cancer (SCLC), Squamous Non-Small-Cell Lung Cancer (sqNSCLC), Triple-Negative Breast Cancer (TNBC), Upper Tract Urothelial Cancer (UTUC), Urothelial Carcinoma (UC or TCC), or Uveal Melanoma (UM).

[0033] The present application further includes, kits for treatment of cancer, including the isolated polypeptides, antibodies, antigen binding antibody fragments, multispecific antibodies, or pharmaceutical compositions described above and instructions for using the isolated polypeptide, the antibody or antigen binding antibody fragment, multi-specific antibody or pharmaceutical composition for treatment of cancer or any of the specific types of cancers listed above.

[0034] In further aspects, the present application provides for the use of the isolated polypeptides, antibodies, antigen binding antibody fragments, multispecific antibodies or pharmaceutical compositions described above for the manufacture of a medicament. In some embodiments, the medicament is for treatment of cancer or any of the specific types of cancers listed above.

[0035] In a further aspect, the application provides for the use of the isolated polypeptides, antibodies, antigen binding antibody fragments, multispecific antibodies or pharmaceuticalBIAT-1039WO PATENT APPLICATIONcompositions having both pH-dependent conditionally active B7-H3 antigen binding activity and pH-dependent conditionally active CD3 antigen binding activity for the manufacturing of a medicament for the treatment of cancer or any of the specific types of cancers listed above.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic structure of a bi-valent multi-specific antibody that is a hetero-dimer with one arm for binding to an antigen (Ag) here B7-H3 (CD276) and the other arm for binding to CD3 (CD3).

[0037] Figure 2 is a schematic structure of a tetra-valent multi-specific antibody that is a homodimer with each arm having a binding site to an antigen (Ag), here B7-H3 (CD276) and a binding site to CD3 (CD3).

[0038] Figures 3A-3B show binding activities to human-B7-H3-his recombinant protein by bispecific antibodies at pH 6.0 and pH 7.4, respectively, measured at OD450 nm. Serially diluted bispecific antibodies WT-B7-H3 x CD3, DualCAB-CPSll x CD3-BF45 and DualCAB-CPSl 1 x CD3-BF46, were added to human B7-H3-his recombinant protein coated ELISA plates and binding was detected using anti-human IgG antibody HRP secondary antibody at an absorbance of 450 nm. Binding results at pH 6.0 (Fig. 3A) and at pH 7.4 (Fig. 3B) are presented.

[0039] Figure 4A-4B show binding activities to human-CD38 / 5-heterodimer by bispecific antibodies at pH 6.0 and pH 7.4 using a sandwich assay. Serially diluted bispecific antibodies WT-B7-H3 x CD3, DualCAB-CPSll x CD3-BF45 and DualCAB-CPSl l x CD3-BF46, were added to human CD3s / 5 heterodimer coated ELISA plates, bound, and washed to remove unbound anti-CD3 antibody. Human B7-H3 ECD fused to mouse Fc protein was added, incubated, and unbound Fc protein removed. Binding was detected using anti-mouse IgG antibody HRP secondary antibody at an absorbance of 450 nm. Binding results at pH 6.0 (Fig. 4A) and at pH 7.4 (Fig. 4B) are presented.

[0040] Figure 5A-5B show binding activities to human CD3 s / 5-heterodimer by bispecific antibodies at pH 6.0 and pH 7.4 using a sandwich assay. Serially diluted bispecific antibodies WT-B7-H3 x CD3, DualCAB-CPSll x CD3-BF45 and DualCAB-CPSll x CD3-BF46, were added to human CD3e / 5 heterodimer coated ELISA plates, bound, and washed to remove unbound CD3. Cyno B7-H3 ECD fused to mouse Fc protein was added, incubated, and unbound Fc protein removed. Detection of bound Fc protein was performed using anti-mouse IgG antibody HRP secondary' antibody at an absorbance of 450 nm. Binding results at pH 6.0 (Fig. 5A) and at pH 7.4 (Fig. 5B) are presented.

[0041] Figure 6 shows binding of WT-B7-H3 x CD3 antibody, DualCAB-CPSll x CD3-BF45 antibody and related antibodies anti-PD-Ll; anti-PD-L2; anti-B7-l, anti-B7-2, and anti-B7-H2, to human B7-H3-his antigen.BIAT-1039WO PATENT APPLICATION

[0042] Figure 7 is a bar graph of the binding of the WT-B7-H3 x CD3 and DualCAB-CPSl 1 x CD3-BF45 to human. cyno, rat and mouse B7-H3 at pH 6.0 and pH 7.4.

[0043] Figure 8A-B are graphs of the binding affinities of DualCAB CPS 11 BF45 (Fig. 8A) and DualCAB CPS 11 BF46 (Fig. 8B) at pH 6.0 and pH 7.4 and three different concentrations, to human CD3 using Jurkat cells that express human CD3.

[0044] Figure 9A-B are graphs of the binding affinities of DualCAB CPS 11 BF45 (Fig. 9A) and DualCAB CPS 11 BF46 (Fig. 9B) at pH 6.0 and pH 7.4 and three different concentrations, to human B7-H3 using A375 cells that express human B7-H3.

[0045] Figure 5A-B are graphs of the binding affinities of DualCAB CPS 11 BF45 (Fig. 10A) and DualCAB CPS 11 BF46 (Fig. 10B) at pH 6.0 and pH 7.4 and three different concentrations to human B7-H3 using Detroit562 cells that express human B7-H3.

[0046] Figure 11A-B are graphs of the binding affinities of DualCAB CPS 11 BF45 at pH 6.0 (Fig.11 A) and DualCAB CPS11 BF45 at pH 7.4 (Fig. 11B) to human B7-H3 using A375 cells that express human B7-H3, compared to the binding affinities to human B7-H3 of the wild ty pe B7-H3 / CD3 and isotype / Wt CD3 antibodies under the same conditions.

[0047] Figure 12A-12B are graphs of the binding affinities of DualCAB CPS 11 BF45 at pH 6.0 (Fig. 12A) and DualCAB CPS11 BF45 at pH 7.4 (Fig. 12B) to human B7-H3 using Detroit 562 cells that express human B7-H3, compared to the binding affinities to human B7-H3 of the wild ty pe B7-H3 / CD3 and isotype / Wt CD3 antibodies under the same conditions.

[0048] Figure 13 is a graph of the effects of IV administration of DualCAB CPS 11 BF45 on the growth of tumors in vivo in an A375 CDX humanized mouse model.

[0049] Figure 14 is a graph of the effects of IV administration of DualCAB CPS 11 BF45 on the grow th of tumors in vivo in a Detroit 562 CDX humanized mouse model.DEFINITIONS

[0050] In order to facilitate understanding of the examples provided herein, certain frequently occurring methods and / or terms w ill be defined herein.

[0051] In connection with a measured quantity, the term “about” as used herein refers to the normal variation in that measured quantity that would be expected by a skilled person making the measurement and exercising a level of care commensurate with the obj ective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated, “about” refers to a variation construed in light of the number of reported significant digits and by applying ordinary' rounding techniques.

[0052] The term “affinity” as used herein refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partnerBIAT-1039WO PATENT APPLICATION(e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary7aspects for measuring binding affinity7are described in the following.

[0053] The term “amino acid” as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (— COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds. The “twenty naturally encoded polypeptide-forming alphaamino acids” are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), try ptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0054] The term “antibody” as used herein refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab'. (Fab'ty, Fv, and single chain antibody (SCA) fragments, that are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen (e.g. , a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., (see, e.g., Greenfield (2012) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, ISBN:9781936113811), and are described further, as follows. Antibodies useful in practice may be IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, sig A, IgD or IgE. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography. Various other uses of such antibodies are to diagnose and / or stage disease (e.g.. neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

[0055] The term “antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2: diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.BIAT-1039WO PATENT APPLICATION

[0056] An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzy me papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

[0057] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Tw o Fab' fragments are obtained per antibody molecule treated in this manner.

[0058] An (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.

[0059] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

[0060] The term “antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH. F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0061] The term “single chain Fv” (“scFv”) as used herein is a covalently linked VH: : VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. "dsFv” is a VH::VL heterodimer stabilized by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

[0062] The term “conditional activity” or “conditionally active” refers to an activity, affinity or avidity of an antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment, or multispecific antibody or fragment thereof, that is higher at one or more aberrant conditions as compared to at a normal physiological condition. This conditionally active antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment can exhibit activity in selected regions of the body and / or exhibits increased or decreased activity under aberrant, or permissive, physiological conditions. In one aspect, the conditionally active antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment is virtually inactive at a normal physiological condition but is active at the aberrant condition. For example, in one aspect, the conditionally active antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment is virtually inactive at a normal physiological pH, but is active at lower pH in a tumor microenvironment. In another aspect, the conditionally active antigen binding polypeptide,BIAT-1039WO PATENT APPLICATIONantigen binding antibody, or antigen binding antibody fragment, may be reversibly or irreversibly inactivated at the normal physiological pH. In an additional aspect, the conditionally active antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment, may be reversibly inactivated at the normal physiological pH. In a further aspect, the conditionally antigen binding polypeptide, antigen binding antibody, or antigen binding antibody fragment is derived from a therapeutic protein. In another aspect, the conditionally active multi-specific antibody is used as a drug, or therapeutic agent.

[0063] The term “pH-dependent conditionally active antigen binding activity” as used herein, refers to a higher antigen binding activity under one pH environment compared to the antigen binding activity' to the same antigen in a different pH environment. In an aspect, the conditionally active antibody is less active in normal physiological pH 7.2 to 7.8. or 7.4, but more active under an acidic pH 5.8 to 7.0, or 6.0 to 6.8, or 6.0, for example that exists in a tumor microenvironment. In aspects, the binding activity is measured using known methods at a normal physiological pH of 7.4 and an acidic pH of 6.0 and thus the normal physiological pH of this embodiment is 7.4 and the aberrant pH of this embodiment is the acidic pH of 6.0. The present application provides for and includes pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen. Also included are multispecific antibodies, or antigen binding fragments thereof, comprising a pH-dependent conditionally active antigen binding activity to B7-H3 and CD3.

[0064] The term “full length antibody,” “intact antibody,” or “whole antibody” refers to an antibody which comprises an antigen-binding variable region (VH or VL) as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different “classes”. There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGL IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0065] The term “Fc region” as used herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226. or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specifiedBIAT-1039WO PATENT APPLICATIONherein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al.. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Sendee, National Institutes of Health, Bethesda, Md., 1991.

[0066] The term “variable region” or “variable domain” as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology7, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or Vi, domain from an antibody that binds the antigen to screen a library of complementary7VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.

[0067] The term “framework” or “FR” as used herein refers to variable domain residues other than complementarity determining regions (CDRs or Hl -3 in the heavy chain and LI -3 in the light chain) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1 -H 1 (L 1 )-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0068] The term “binding” as used herein refers to interaction of the variable region or an Fv of an antibody with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure rather than to proteins generally. As used herein, the term “specifically binding” or “binding specifically” means that an antibody variable region or Fv binds to or associates with more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen than with other proteins. For example, an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens. For another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity than it does to related proteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally7found in humans). However, “specifically binding” does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term “selective binding”. In one example, “specific binding” of an antibody variable region or Fv (or other binding region)BIAT-1039WO PATENT APPLICATIONbinds to an antigen, means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100x1 O’9M (nM) or less, such as 50 nM or less, for example 20 nM or less, such as, 15 nM or less, or 10 nM or less ,or 5 nM or less, 2 nM or less, or 1 nM or less.

[0069] A single chain antibody (“SCA”) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide liner, and which may include additional amino acid sequences at the amino-and / or carboxyl- termini. As an example, an scFv antibody is a single-chain antibody. For example, a single chain antibody may include a tether segment for linking to the encoding polynucleotide. A functional single chain antibody generally contains a sufficient portion of the variable region of a light chain and a sufficient region of the variable region of a heavy chain so as to retain the property of a full-length antibody for binding to a specific target molecule or epitope. Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and THE. Rabbits, eds., (1989) Academic press: San Diego, Calif., pp. 361-368), most frequently encoded by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human heavy chain or light chain gene sequence. A functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property' of binding to a specific target molecule, typically a receptor or antigen (epitope).

[0070] The term "diabodies" as used herein refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy -chain variable domain (VH) connected to a lightchain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0071] The term “avidity7’ as used herein refers to the combined strength of multiple binding sites between two molecules, such as between multiple antigen binding sites of a multi-specific antibody simultaneously interacting with two targets. When more than one binding interaction is present, the two molecules will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, thereby providing a greater effective total binding strength (avidity7) compared to the strength of binding of the individual binding sites (affinity7). Thus, the avidity7is related to both the affinity' of individual binding site and specific epitopes, and also the valence of the multi-specific antibody and the antigen. For example, the interaction between a bispecific antibody and an antigen with a repeating epitope structure, such as a polymer, would be one of high avidity because the binding can be to multiple epitopes on the antigen.BIAT-1039WO PATENT APPLICATION

[0072] The terms “treatment,” “treat,” or “treating” as used herein refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the application are used to delay development of a disease or to slow the progression of a disease, reducing the number of symptoms or reducing the severity, duration, or frequency of one or more symptoms of disease or disorder (e.g., a cancer) in a subject. The term treating can also mean delaying the onset or progression of symptoms, or progression of severity of symptoms, of the disorder or disorder in a subject, or increasing the longevity of a subject having the disorder or disorder.

[0073] The term “tumor” as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0074] The terms “cancer” and “cancerous” as used herein refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to B-cell lymphomas (Hodgkin's lymphomas and / or non-Hodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, adenocarcinoma, glioma, melanoma, lymphoma, cystadenoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer. In aspects, the cancer can be a B7-H3 (CD276) antigen expressing cancer. Examples of B7-H3 (CD276) expressing cancers include hepatocellular Carcinoma (HCC), Pancreatic Adenocarcinoma (PAC), Pancreatic Adenocarcinoma (PAC), Colorectal Cancer (CRC), Cervical Cancer (CC), Endometrial Cancer (EC), Ovarian Cancer (OC), Breast Cancer (BC), Breast Invasive Carcinoma (TCGA), Renal Cell Carcinoma (RCC), Prostate Cancer (PC), Lung Cancer. Liver cancer, squamous cell carcinoma, basal cell carcinoma, Neuroblastoma, Melanoma, Glioblastoma multiforme, a carcinoid tumor, and pediatric retinoblastoma.

[0075] An “individual,” “patient” or “subject” is a human or an animal. For example, the subject may be a mammal selected from domesticated animals (eg., cows, sheep, cats, dogs, and horses),BIAT-1039WO PATENT APPLICATIONprimates (e.g, humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0076] The term “multi-specific antibody” as used herein is a full-length antibody, an antibody fragment or a construct comprising one or more full-length antibodies and antibody fragments, which has at least two different binding sites each capable of binding to an epitope on the same or different antigen. The construct may be engineered antibodies with two, three or more (e.g. four, five, six, or seven) functional antigen binding sites are also encompassed within the scope of the multi-specific antibody (see, e.g., US 2002 / 0004587 Al and Brinkman and Kontermann, MAbs, vol. 9, pp. 182-212, 2017).

[0077] The term “pharmaceutically acceptable salt” as used herein refers to a salt form of the conditionally active antigen binding polypeptide, antigen binding antibody, antigen binding antibody fragment, or multi-specific antibody of the present application. The salt form may be acid addition salts (e.g., formed with free amino groups) and which are formed with inorganic acids such as hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.

[0078] The term “preventing” as used herein refers to avert or avoid a condition from occurring. In some aspects, preventing is directed to use the antigen binding polypeptide, antigen binding antibody, antigen binding antibody fragment, or multi-specific antibody of the present application to ameliorate the damage associated with a condition, such as cancer.

[0079] The term “therapeutically effective amount” as used herein means any amount of the antigen binding polypeptide, antigen binding antibody, antigen binding antibody fragment, or multi-specific antibody of the present application, which, as compared to a corresponding subject who has not received such amount, results in, but is not limited to, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function as well as amounts effective to cause a physiological function in a patient which enhances or aids in the therapeutic effect of a second pharmaceutical agent.

[0080] The term “human antibody” as used herein is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.BIAT-1039WO PATENT APPLICATION

[0081] The term ‘‘humanized” antibody as used herein refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0082] The term “pharmaceutical formulation” as used herein refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0083] The term “pharmaceutically acceptable carrier” as used herein refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject., A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0084] The terms “purified” and “isolated” used herein when referring to an antibody or polypeptide according to the application or to a nucleotide sequence, indicates that the molecule is present in the substantial absence of other biological macromolecules of the same type. The term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present. An “isolated” nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition. For review of methods for assessment of antibody and polypeptide purity', see, e.g.. Flatman et al., J. Chromatogr. B, 848:79-87 (2007).DETAILED DESCRIPTION

[0085] For illustrative purposes, the principles of the present application are described by referencing various exemplary' aspects. Although certain aspects of the application are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be employed in, other systems and methods. Before explaining the disclosed aspects of the present application in detail, it is to be understood that the application is not limited in its application to the details of any particular aspect shown. Additionally, the terminology7used hereinBIAT-1039WO PATENT APPLICATIONis for the purpose of description and not for limitation. Furthermore, although certain methods are described with reference to steps that are presented herein in a certain order, in many instances, these steps can be performed in any order as may be appreciated by one skilled in the art; hence the described method is therefore not limited to the particular arrangement of steps disclosed herein.

[0086] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Furthermore, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. The terms “comprising,” “including,” “having,” and “constructed from” can also be used interchangeably herein.

[0087] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratios, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about,” whether or not the term “about” is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present application. At the very7least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by7applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0088] It is to be understood that each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every7other component, compound, substituent, or parameter disclosed herein.

[0089] It is also to be understood that each amount / value or range of amounts / values for each component, compound, substituent, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount / value or range of amounts / values disclosed for any other component(s), compounds(s), substituent(s), or parameter(s) disclosed herein and that any combination of amounts / values or ranges of amounts / values for two or more component(s), compounds(s), substituent(s), or parameters disclosed herein are thus also disclosed in combination with each other for the purposes of this description.

[0090] It is further understood that each lower limit of each range disclosed herein is to be interpreted as disclosed in combination with each upper limit of each range disclosed herein for the same component, compounds, substituent, or parameter. Thus, a recitation of two ranges is to be interpretedBIAT-1039WO PATENT APPLICATIONas a recitation of four ranges derived by combining each low er limit of each range with each upper limit of each range. A recitation of three ranges is to be interpreted as a recitation of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, specific amounts / values of a component, compound, substituent, or parameter disclosed in the description or an example is to be interpreted as a recitation of either a low er or an upper limit of a range and thus can be combined with any other lower or upper limit of a range or specific amount / value for the same component, compound, substituent, or parameter disclosed elsewhere in the application to form a range for that component, compound, substituent, or parameter.Isolated anti-B7-H3 (CD276) polypeptides

[0091] The present application relates to an isolated polypeptide having a pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen, the polypeptide comprising:a heavy chain variable region comprising three complementarity determining regions (CDRs) having amino acid sequences Hl, H2, and H3, wherein:the Hl sequence is GFDFSSFGEH (SEQ ID NO: 1);the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NO:2); andthe H3 sequence is GRENIYYGARLDY (SEQ ID NO:3); anda light chain variable region including three complementarity' determining regions having amino acid sequences LI, L2, and L3, wherein:the LI sequence is KASQNVDDQVA (SEQ ID NO:4);the L2 sequence is SASYRYS (SEQ ID NO: 5); andthe L3 sequence is QQYNKYPFT (SEQ ID NO:6);wherein the isolated polypeptide has a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0092] In aspects, the present application provides for an isolated polypeptide having a pH-dependent conditionally active antigen binding activity' to the B7-H3 antigen from Homo sapiens (Genbank Gene ID:80381) and to the B7-H3 antigen from Cynomolgus fascicularis (Genbank Gene ID: 102131295).

[0093] In aspects of the present application, the light chain variable region that includes the CDRs having the sequences LI, L2 and L3 comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In some embodiments, the framework regions of these light chain variable regions can vary while the CDRs remain the same. In certain embodiments, theBIAT-1039WO PATENT APPLICATIONchanges to the non-CDR regions of the light chain variable region are conservative amino acid substitutions. Thus, in one embodiment, the light chain variable region includes the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. Similarly, in another embodiment, the light chain variable region includes the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In yet another embodiment, the light chain variable region the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In yet another embodiment, the light chain variable region includes the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In yet another embodiment, the light chain variable region includes the CDRs having the sequences LI, L2 and L3 and has an amino acid sequence that is identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16. and SEQ ID NO: 17. In each embodiment, the light chain variable region retains the anti-B7-H3 pH-dependent conditional binding activity.

[0094] In other aspects of the present application the heavy chain variable region of the isolated polypeptide having a pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276), comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 18, and retains the anti-B7-H3 pH-dependent conditional binding activity. More specifically, in aspects, the framework regions can vary while the CDRs remain the same. In certain embodiments, the changes to the non-CDR regions of the heavy chain variable region are conservative amino acid substitutions. Thus, in one embodiment, the heavy chain variable region includes the CDRs having the sequences Hl, H2 and H3 and has an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. Similarly, in another embodiment, the heavy chain variable region includes the CDRs having the sequences Hl, H2 and H3 and has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In yet another embodiment, the heavy chain variable region includes the CDRs having the sequences Hl, H2 and H3 and has an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In yet another embodiment, the heavy chain variable region includes the CDRs having the sequences Hl, H2 and H3 and has an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16,BIAT-1039WO PATENT APPLICATIONand SEQ ID NO: 17. In yet another embodiment, the heavy chain variable region includes the CDRs having the sequences Hl. H2 and H3 and has an amino acid sequence that is identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. In each embodiment, the heavy chain variable region retains the anti-B7-H3 pH-dependent conditional binding activity7.

[0095] In aspects, the isolated polypeptide having a pH-dependent conditionally active antigen binding activity to B7-H3 (CD276) has a ratio of B7-H3 (CD276) antigen binding activity at pH 6.0 to B7-H3 (CD276) antigen binding activity at pH 7.4 of at least 1.3 to 1 (1.3: 1). In other aspects, the ratio of the B7-H3 (CD276) antigen binding activity' at pH 6.0 to the B7-H3 (CD276) antigen binding activity7at pH 7.4 is at least 1.5 to 1 or from 2: 1 to 20: 1. In yet further aspects, wherein the ratio of the B7-H3 (CD276) antigen binding activity7at pH 6.0 to the B7-H3 (CD276) antigen binding activity7at pH 7.4 is from 2:1 to 50:1. In aspects, the pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) at pH 6.0 or at pH 7.4 is reversible.

[0096] The present application provides for, and includes, isolated polypeptides having pH-dependent antigen binding activity to a B7-H3 (CD276) antigen, wherein the isolated polypeptides are configured as an antibody or an antigen binding antibody fragment thereof.

[0097] In some aspects, the isolated polypeptide further includes a binding activity to CD3 antigen which binding activity' to CD3 antigen may be a pH-dependent conditionally active antigen binding activity to CD3 antigen. In embodiments with a pH-dependent conditional binding activity to CD3, the light chain variable region further includes six anti-CD3 complementarity determining regions having sequences L4, L5, L6, L7, L8, and L9, wherein: the L4 sequence is GFTFNTYAMN (SEQ ID NO:1), the L5 sequence is RIRSKYNNYATYYADSVKD (SEQ ID NO:8), the L6 sequence is HSNFGNSKVSWFAY (SEQ ID NO:9) or HTNFGNSKVSWFAY (SEQ ID NOTO), the L7 sequence is RSSAGAVTTSNYDN (SEQ ID NO: 11), the L8 sequence is GTNKRAP (SEQ ID NO: 12), and the L9 sequence is ALWYSNLWV (SEQ ID NO: 13). The light chain variable region including the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8, and L9 binds to CD3 antigen and as a result the isolated polypeptide has a higher binding activity to the CD3 antigen at pH 6.0 than at pH 7.4.

[0098] In some aspects, the isolated poly peptide has a ratio of CD3 antigen binding activity at pH 6.0 to CD3 antigen binding activity at pH 7.4 of at least 1.3 to 1. In some aspects, the isolated polypeptide has a ratio of the CD3 antigen binding activity7at pH 6.0 to the CD3 binding activity7at pH 7.4 of at least 1.5:1 or from 2:1 to 20:1.

[0099] In some aspects, the isolated polypeptide that includes the light chain variable region including the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6,BIAT-1039WO PATENT APPLICATIONL7, L8 and L9 is selected from a single chain variable fragment (scFv), a nanobody (Camelid VHH), a DARPIN, a minibody, or a tri-scFv, each having pH-dependent conditional CD3 antigen binding activity.

[0100] In certain aspects, the antibody, or antigen binding antibody fragment is a multispecific antibody or antigen binding antibody fragment. Multispecific antibodies of the present application may have pH-dependent conditionally active antigen binding activity to CD3 antigen and pH-dependent conditionally active antigen binding activity to B7-H3 (CD276) antigen.B. Anti-B7-H3 Antibodies

[0101] In another aspect, the present application relates to a pH-dependent conditionally active anti-B7-H3 antibody or antibody fragment thereof comprising the isolated polypeptides described above and having a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0102] In another aspect, the application relates to a pH-dependent conditionally active anti-B7-H3 antibody or antigen binding antibody fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions, said regions having sequences Hl, H2, and H3, wherein:the Hl sequence is GFDFSSFGEH (SEQ ID NO: 1);the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NO:2); andthe H3 sequence is GRENIYYGARLDY (SEQ ID NO:3); anda light chain variable region including three complementarity determining regions having amino acid sequences LI, L2, and L3, wherein:the LI sequence is KASQNVDDQVA (SEQ ID NO:4);the L2 sequence is SASYRYS (SEQ ID NO: 5); andthe L3 sequence is QQYNKYPFT (SEQ ID NO:6); andwherein the isolated polypeptide has a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0103] In certain aspects the pH-dependent conditionally active anti-B7-H3 antibodies and antigen binding antibody fragments thereof include the combinations of six CDR’s set forth in the list above for the isolated polypeptides.

[0104] The terms '‘anti-B7-H3 antibody”, “B7-H3 antibody”, and ‘'an antibody that binds to B7-H3” as used herein refer to an antibody that is capable of binding to the B7-H3 antigen with sufficient affinity' such that the antibody is useful as a therapeutic agent in targeting B7-H3. The official symbol of B7-H3 is CD276 as designated by the HUGO Gene Nomenclature Committee and the terms CD276 and B7-H3 are used interchangeably herein. The Genbank Gene ID of CD276 is 80381. TheBIAT-1039WO PATENT APPLICATIONB7-H3 protein is a member of the immunoglobulin superfamily and is known to participate in the regulation of T-cell-mediated immune responses.

[0105] In one aspect, the extent of binding of an anti- B7-H3 antibody to an unrelated, non- B7-H3 protein is less than about 10% of the binding of the antibody to B7-H3 as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to B7-H3 has a dissociation constant (Kd) for binding to B7-H3 of - IxlO'6M (pM), - lOOxlO’9M (nM), - 10 nM, - 1 nM, -0.1 nM, -0.01 nM, or -0.001 nM (e.g. 10‘8M) or less. e.g. from 10’8M to I0’13M, e.g., from 10’9M to 10’13M).

[0106] In certain aspects, an anti-B7-H3 antibody binds to an epitope of B7-H3 that is conserved among B7-H3 from different species, for example, the extracellular domain of B7-H3.

[0107] In certain aspects the specification provides an antibody or antibody fragment that specifically binds to B7-H3 protein, said antibody or antibody fragment comprising a heavy chain variable region that may have the amino acid sequence of SEQ ID NO: 15, and the light chain variable region may have the amino acid sequence of SEQ ID NO: 14., w herein said antibody has a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

[0108] In aspects of the present application, the pH-dependent conditionally active anti-B7-H3 antibodies and antigen binding antibody fragments thereof comprise a light chain variable region that includes the CDRs having the sequences LI, L2 and L3 and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17. More specifically, in aspects, the framework regions of the light chain variable region can vary while the CDRs remain the same. In aspects, the changes to the regions outside the CDRs are conservative amino acid substitutions.

[0109] In other aspects of the present application, the pH-dependent conditionally active anti-B7-H3 antibodies and antigen binding antibody fragments thereof include a the heavy chain variable region of the isolated polypeptide having a pH-dependent conditionally active antigen binding activity to B7-H3 (CD276), includes an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 18 and retains anti-B7-H3 pH conditional binding activity.

[0110] The antibody or antigen-binding antibody fragment thereof of this aspect may have a ratio of binding affinity to the B7-H3 protein at a pH in a tumor microenvironment to a binding affinity to the B7-H3 protein at a different pH in anon-tumor microenvironment of at least about 1.3:1, at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1. at least about 8:1, at least about 9:1, at least about 10:1. at least about 20:1. at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.BIAT-1039WO PATENT APPLICATION[OHl] In aspects of the present application, the pH-dependent conditionally active anti-B7-H3 antibodies and antigen binding antibody fragments comprise a light chain variable region that includes the CDRs having the sequences LI, L2 and L3 comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17 and a the heavy chain variable region of the isolated polypeptide having a pH-dependent conditionally active antigen binding activity to B7-H3 (CD276), includes an amino acid sequence that is at least 90%, at least 95%. at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 15, and SEQ ID NO: 18. More specifically, in aspects, the framework regions can vary while the CDR regions remain the same. In aspects, the changes to the regions outside the CDRs are conservative amino acid substitutions.

[0112] The heavy chain variable regions and the light chain variable regions of the present application were each obtained from a parent antibody using a method disclosed in U.S. Patent No.8,709,755. This method of generating the heavy chain variable regions and the light chain variable regions, as well as the method of generating antibodies and antigen binding antibody fragments disclosed in U.S. Patent No. 8,709,755. are hereby incorporated by reference herein.

[0113] The pH-dependent conditionally active anti-B7-H3 antibodies and antigen binding antibody fragments including these heavy and light chain variable regions can specifically bind to B7-H3 protein, especially human and cynomolgus monkey B7-H3 protein. Antibodies or antibody fragments thereof comprising a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have higher binding affinity to B7-H3 protein at a pH in the tumor microenvironment (e.g. pH 5.0-7.0 or pH 6.0) than at a pH in a non-tumor microenvironment (e.g. pH 7.2-7.8 or pH 7.4). As a result, these anti- B7-H3 antibodies or antibody fragments have a higher binding affinity to B7-H3 protein in a tumor microenvironment in comparison with their binding affinity to B7-H3 protein in a typical normal tissue microenvironment.

[0114] Anti-B7-H3 antibodies or antigen binding antibody fragments of the present specification are thus expected to exhibit reduced side-effects, relative to non-conditionally active anti-B7-H3 antibodies, due to their reduced binding affinity to B7-H3 protein in the normal tissue microenvironment. Anti- B7-H3 antibodies or antibody fragments of the present application are also expected to have a comparable or greater efficacy than monoclonal anti-B7-H3 antibodies known in the art. Several examples of anti-B7-H3 antibodies that exhibited essentially no side effects and comparable or greater efficacy than an isotype control antibody are demonstrated in Example 10 of this application in in vivo testing in an A375 CDX humanized mouse model and a Detroit562 CDX humanized mouse model. Exemplary anti-B7-H3 antibodies in cynomolgus monkeys in Example 11BIAT-1039WO PATENT APPLICATIONbelow presented no related effects in clinical observations, body weights, food consumption, clinical pathology parameters, peripheral blood immunophenotyping. or anatomic pathology findings providing a strong indication of reduced side effects due to binding to normal tissue. This combination of features permits use of a higher dosage of these anti-B7-H3 antibodies or antigen binding antibody fragments thereof and / or a longer duration of therapy due to their reduced side effects, which may provide a more effective therapy option.

[0115] In addition to the polypeptides and the antibodies or antibody fragments having the heavy chain variable regions and light chain variable regions described, the present application also includes variants of these polypeptides, antibodies and antibody fragments, that can specifically bind to B7-H3 protein, especially human and cynomologus B7-H3 protein. In some aspects, these variants have different framework sequences. In other aspects, the portion of the amino acid sequence of the heavy and light chain variable regions outside of the complementarity determining regions may be mutated in accordance with the principles of substitution, insertion and deletion, as discussed in this application to provide these variants. In still further aspects, the constant regions may be modified to provide these variants. In still further aspects, two or all three of these regions may be modified to provide these variants.

[0116] In deriving these variants, one is guided by the process as described herein. The variants of the heavy chain and light chain variable regions may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the heavy and light chain variable regions, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the heavy and light chain variable regions. Any combination of deletion, insertion, and substitution can be made to arrive at the antibodies or antibody fragments of the present application, provided that they possess the desired characteristics, e.g. antigen-binding to human and cynomolgus B7-H3 protein and pH-dependent conditional activity.C. Substitution, Insertion, and Deletion Variants

[0117] In certain aspects, antibody or antibody fragment variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions (FRs). Conservative substitutions are shown in Table 2 under the heading of “conservative substitutions.” More substantial changes are provided in Table 2 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody or antibody fragment of interestBIAT-1039WO PATENT APPLICATIONand the products screened for a desired activity, e.g., retained / improved antigen binding, conditional activity and / or decreased immunogenicity.Table 2: Amino acid substitutions

[0118] Additional details on substitutions, insertions, and deletion variants according to the present application are known in the art. For example, International Patent Publication No. WO 2021 / 151020, published July 29, 2021, hereby incorporated by reference in its entirety.BIAT-1039WO PATENT APPLICATIONMultispecific Antibodies and Antibody Fragments

[0119] The application also provides multi-specific pH conditional binding anti-B7-H3 antibodies, e.g. bispecific antibodies. Multi-specific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. The other antigen bound by the multi-specific antibodies of the present application is preferably the tumor-reactive lymphocyte antigen, CD3, which is an antigen of a lymphocyte that targets tumor cells. Particularly, the lymphocyte is tumor reactive when the lymphocyte can attack, inhibit or destroy tumor cells. The tumor-reactive lymphocyte may be selected from T cells, macrophages. Jurkat cells, monocytes. NK cells, activated NK. cells, neutrophils, eosinophils, basophils, B- cells, and lymphokine-activated killer (LAK) cells. The T cells may be naive T cell, ahelperT cell, an effector T-cell, amemoiy T-cell, a cytotoxic T cell, an antigenspecific T-cell, and a CD28-CD27-CD4 positive T-cell.

[0120] Without wishing to be limited by theory, the antigen binding polypeptide, antigen binding antibody, antigen binding antibody fragment thereof, or multi-specific antibody of the present application binds to both the target cell and tumor-reactive lymphocyte to thereby bring the target cell in close proximity to the tumor-reactive lymphocyte. This is believed to facilitate an attack by the tumor-reactive lymphocyte on the target cell to thereby inhibit, damage or destroy the target cell. A therapeutic effect of inhibition or removing tumor cells may be achieved by using the antigen binding polypeptide, antigen binding antibody, antigen binding antibody fragment thereof, or multispecific antibody of the present application to bring the reactive lymphocyte to tumor cells for inhibition, destruction and removal of the tumor cells from the subject.

[0121] The terms “anti-CD3 polypeptide”. “anti-CD3 antibody”. “CD3 antibody”, "an antibody that binds to CD3”, andC'anti-CD3 scFV”, as used herein refer to a polypeptide or antibody that is capable of binding CD3 with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting CD3. In one aspect, the extent of binding of an anti- CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3 as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody or polypeptide that binds to CD3 has a dissociation constant for binding to CD3 (Kd) of -IxlO’6M (pM), -lOOxlO"9M (nM), ~10 nM, ~1 nM, -0.1 nM, -0.01 nM, or -0.001 nM (e.g. 10‘8M or less, e.g. from 10‘8M to 10‘13M, e.g., from 10‘9M to IO"13M). In certain aspects, an anti-CD3 antibody or polypeptide binds to an epitope of CD3 that is conserved among CD3 from different species.

[0122] In certain aspects, bispecific conditionally active antibodies may bind to two different epitopes of B7-H3. The multi-specific antibody binds to both B7-H3 and the CD3 antigen with a greater activity', affinity and / or avidity' at a pH of 6.0 than at a pH of 7.4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.BIAT-1039WO PATENT APPLICATION

[0123] In some aspects, the binding of the multi-specific antibody to the B7-H3 antigen and / or tumor-reactive lymphocyte CD3 antigen is reversible. Reversible binding indicates that the multispecific antibody may bind to the B7-H3 antigen and / or tumor-reactive lymphocyte CD3 antigen, followed by separation of the two. The separated multi-specific antibody is capable of binding to the B7-H3 antigen and tumor-reactive lymphocyte CD3 antigen again. The reversibility of the binding can be triggered by, for example, a change in the pH to which the bispecific antibody is exposed.

[0124] In some aspects, the B7-H3 antigen is an antigen preferentially associated with the target cell but is less prevalent with other cell ty pes. In this manner, the multi-specific antibody of the present application can preferentially interact with the target B7-H3 expressing cell. The target cell may be a cancer cell.

[0125] In aspects, the targeted cancer cell is a B7-H3 expressing cancer cell. Exemplary cancer cells include, but are not limited to Carcinoma (CA), Castration-Resistant Prostate Cancer (CRPC), Central Nervous System (CNS) / Leptomeningeal Metastases (LM), Clear Cell Carcinoma (CCC) of the ovary, Colorectal Cancer (CRC), Epithelial Ovarian Cancer (EOC), Esophageal Squamous Cell Carcinoma (ESCC), Head And Neck Squamous Cell Carcinoma (HNSCC), Head And Neck Squamous Cell Carcinoma (HNSCC), Lentigo malignant Melanoma (LMM), Medulloblastoma (MB), Melanoma, Metastatic Castration-Resistant Prostate Cancer (mCRPC), Muscle Invasive Bladder Cancer (MIBC), Neuroblastoma (NBL), Non-Muscle Invasive Bladder Cancer (NMIBC), Non-Small Cell Lung Cancer (NSCLC), Prostate Cancer (PCa), Renal Cell Carcinoma (RCC), Retinoblastoma (Rb). Rhabdomyosarcoma (RMS), Small Cell Lung Cancer (SCLC), Squamous Non-Small-Cell Lung Cancer (sqNSCLC), Triple-Negative Breast Cancer (TNBC), Upper Tract Urothelial Cancer (UTUC), Urothelial Carcinoma (UC or TCC), and Uveal Melanoma (UM).

[0126] The structure / format of the multi-specific antibody may be any one of the structures / formats described in Brinkmann and Kontermann, "‘The making of bispecific antibodies, " MABs. vol. 9, pp.182-212, 2017, or as described in Orcutt et al., Protein Engineering, Design & Selection, 23(4): 221-228 (2010). Specifically, Figure 2 of Brinkmann and Kontermann describes 19 different structures / formats for bispecific antibodies. These structures / formats include: (1) bispecific antibody conjugates; (2) hybrid bispecific IgG2; (3) “variable domain only” bispecific antibody molecules; (4) CH1 / CL fusion proteins; (5) Fab fusion proteins; (6) non-immunoglobulin fusion proteins; (7) Fc-modified IgGs: (8) appended and Fc-modified IgGs; (9) modified Fc and CH3 fusion proteins; (10) appended IgGs-HC fusions; (11) appended IgGs-LC fusions; (12) appended IgGs-HC&LC fusions; (13) Fc fusions; (14) CH3 fusions; (15) IgE / IgM CH2 fusions; (16) F(ab’)2 fusion; (17) CH1 / CL fusion proteins; (18) modified IgGs; and (19) non-immunoglobulin fusions. In one embodiment, the muli-specific antibody of the present application is an appended IgGs-LC fusion. Similarly, OrcuttBIAT-1039WO PATENT APPLICATIONdescribes bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. The structure of the heavy chain, light chain, and fully assembled bsAB with N- and C-termini indicated, is shown in Figure 1 of Orcutt.

[0127] Additional techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello. Nature, vol. 305, pp. 537-540, 1983), WO 93 / 08829. and Traunecker et al., EMBOJ. vol. 10, pp. 3655-3659, 1991), and '‘knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,1 8). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g.. U.S. Pat. No. 4.676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single-chain Fv (scFv) dimers (see, e.g. Gruber et al., J. Immunol., vol. 152, pp. 5368-5374, 1994); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991.

[0128] Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,’' are also included herein (see, e.g. US 2006 / 0025576A1).

[0129] The anti-B7-H3 antibodies or antibody fragments of the application may be produced using recombinant methods and compositions, which are described in detail in US 2016 / 0017040.

[0130] The physical / chemical properties and / or biological activities of the anti-B7-H3 antibodies or antibody fragments of the application may be tested and measured by various assays known in the art. Some of these assays are described in U.S. Patent No. 8,853.369.

[0131] In particular aspects, the multi-specific antibody may be a bivalent scFv-Fc heterodimer or a tetravalent homodimer “butterfly” as shown in Figure 1 and Figure 2, respectively. In these two structures, the Ag represents the pH-dependent conditionally active antigen binding activity to B7-H3 (CD276) and the CD3 refers to a pH-dependent conditionally active antigen binding activity to CD3 antigen. The multi-specific antibodies of Figure 1 and 2 have a first binding site to a cell antigen (Ag- B7-H3), which is linked to a first heavy chain constant region (e.g., IgG) and a second binding site to a reactive lymphocyte antigen (e.g., CD3), which is linked to a second heavy chain constant region (e.g., IgG). The two heavy chains are engineered such that they can only form heterodimers, for example, by using the knob-in-hole technique. The first and second binding sites are scFvBIAT-1039WO PATENT APPLICATIONantibody fragments binding to the cell antigen and reactive lymphocyte antigen, respectively. Both of the first and second binding sites are pH-dependent conditionally active antigen binding sites.

[0132] The multi -specific antibodies of Figure 1 and 2 may have a full-length IgG antibody binding to the cell specific antigen (Ag) and a scFv antibody binding to a reactive lymphocyte antigen (e.g., CD3). The scFv antibody is linked to the C terminus of the light chain of the IgG antibody via a linker. The linker may be a short Alanine linker (Ala)n, a Serine linker (Ser)n, a hydrophilic linker or a glycine-serine-rich linker. The heavy chain of the IgG antibody pairs with the light chain of the IgG antibody that has been linked to the scFv antibody, thus forming half of the homodimer. This multispecific antibody has a “butterfly” configuration.

[0133] In some aspects, the multi-specific antibody comprises an IgG antibody or fragment thereof that binds to a tumor-reactive lymphocyte antigen and a single chain antibody that binds to a tumor cell antigen, also forming a “butterfly” configuration as shown in Figure 2. The single chain antibody may be an scFv antibody. The scFv antibody may be attached to a C terminus of the IgG antibody via a linker as described herein.

[0134] The binding sites of the multi-specific antibody of the application each comprise a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region may be a single chain antibody format or may be a two-chain format as formed by pairing of a light chain and heavy chain. Both binding sites may have pH-dependent conditional binding activity.

[0135] The present application further includes, and provides for, multispecific antibodies having a pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen and further comprising a pH-dependent conditionally active antigen binding activity to CD3 antigen.

[0136] In some aspects, the multi-specific antibody or antibody fragment comprises a heavy chain variable region including three anti-B7-H3 complementarity determining regions, Hl, H2, and H3, wherein:the Hl sequence is GFDFSSFGEH (SEQ ID NO:1);the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NO:2); andthe H3 sequence is GRENIYYGARLDY (SEQ ID NO:3);a light chain variable region including three anti-B7-H3 complementarity determining regions having sequences LI, L2, and L3, wherein:the LI sequence is KASQNVDDQVA (SEQ ID NO:4);the L2 sequence is SASYRYS (SEQ ID NO:5); andthe L3 sequence is QQYNKYPFT (SEQ ID NO:6); andsix anti-CD3 complementarity determining regions, L4, L5, L6, L7, L8, and L9, whereinBIAT-1039WO PATENT APPLICATIONthe L4 sequence is GFTFNTYAMN (SEQ ID NO: 7),the L5 sequence is RIRSKYNNYATYYADSVKD (SEQ ID NO: 8),the L6 sequence is HSNFGNSKVSWFAY (SEQ ID NO: 9) or HTNFGNSKVSWFAY (SEQ ID NOTO,the L7 sequence is RSSAGAVTTSNYDN (SEQ ID NO: 11),the L8 sequence is GTNKRAP (SEQ ID NO: 12), andthe L9 sequence is ALWYSNLWV (SEQ ID NO: 13); andwherein the light chain variable region including the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8, and L9, binds to CD3 antigen and the multispecific antibody has a higher binding activity' to the CD3 antigen at pH 6.0 than at pH 7.4.

[0137] In a binding site that has a conditional activity, one or both of the light chain and heavy chain variable regions may contain a mutation(s) conferring conditional activity. Exemplary light chain variable regions binding to CD3 include a conditionally active light chain variable region with an amino acid sequence of SEQ ID NO: 19 and a conditionally active light chain variable region with an amino acid sequence of SEQ ID NO:20.

[0138] In specific aspects of the application, the multi-specific antibodies or antibody fragments are bi-specific antibodies that bind to B7-H3 and CD3. Such multi-specific antibodies or antibody fragments may be selected from each of the following combinations of a heavy chain variable region and a light chain variable region as set forth below.

[0139] In an aspect, the light chain variable region comprising the six anti-CD3 complementarity’ determining regions having the sequences L4, L5, L6, L7, L8 and L9, is an scFv and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical, to an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO:20. More specifically, in aspects, the framework and linker regions can vary while the CDRs remain the same. In aspects, the changes to the regions outside the CDRs are conservative amino acid substitutions.

[0140] Exemplary light chain variable regions binding to B7-H3 are the light chain variable regions with the amino acid sequences of SEQ ID NO: 14. Exemplary heavy chain variable regions binding to B7-H3 (CD276) include heavy chain variable regions with the amino acid sequences of SEQ ID NO: 15. One of the light chain variable regions is combined with one of the heavy chain variable regions to form a binding site for B7-H3 (CD276). The binding site for B7-H3 (CD276) is linked with a single chain anti-CD3 antibody having an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 19 or SEQ ID NO:20 to form a multi-specific antibody that binds to both B7-H3 (CD276) and CD3.BIAT-1039WO PATENT APPLICATION

[0141] Some examples of multi-specific antibodies that bind to B7-H3 (CD276) and CD3 are shown below in Table 3.Table 3: Multi-Specific Antibodies That bind to B7-H3 (CD276) and CD3

[0142] In an aspect, the present application includes bispecific antibodies and antigen-binding fragments thereof having a pH-dependent conditionally active antigen binding activity to CD3 antigen provided by six anti-CD3 complementarity determining regions linked to the carboxy terminus of the light chain of the pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen. In an aspect, the application includes bispecific antibodies and antigen-binding antibody fragments that have a ratio of CD3 antigen binding activity at pH 6.0 to the CD3 antigen binding activity at pH 7.4 of at least 1.3 to 1. In some aspects, the ratio of the CD3 antigen binding activity at pH 6.0 to the CD3 binding activity at pH 7.4 is at least 1.5: 1 or from 2 to 20. In yet other aspects, the ratio of the CD3 antigen binding activity at pH 6.0 to the CD3 binding activity at pH 7.4 is from 2 to 50. In aspects of the present application, the binding to the CD3 antigen at pH 6.0 is reversible.

[0143] Also provided for and included in the present application are bispecific antibodies and antigen-binding antibody fragments thereof having a pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen and further comprising a pH-dependent conditionally active antigen binding activity to CD3 antigen wherein the light chain variable region comprising the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8 and L9 is selected from a single chain variable fragment (scFv), a nanobody (Camelid VHH). a DARPIN, a minibody, or a tri-scFv, each having conditional CD3 antigen binding activity. In an aspect, the pH-dependent conditionally active antigen binding activity to CD3 antigen is provided by an scFv.

[0144] In aspects according to the present application, the anti-CD3 binding activity is provided by a svFv linked via a linker to the carboxy terminal end of the light chain variable region that includes the CDRs having the sequences LI, L2 and L3. In an aspect, the anti-CD3 scFv comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:17. More specifically, in aspects, the framework and linker regions of the anti-CD3 scFv can vary while theBIAT-1039WO PATENT APPLICATIONCDRs remain the same. In aspects, the changes to the regions outside the CDRs are conser ative amino acid substitutions.Pharmaceutical Formulations

[0145] The multi-specific antibodies of the present application may be included in pharmaceutical compositions, medical devices, kits, or articles of manufacture for therapeutic or prophylactic use. Suitable pharmaceutical compositions, medical devices, kits, or articles of manufacture are described in detail in WO 2016 / 138071, the disclosure of which is incorporated by reference herein for the purpose of describing these suitable pharmaceutical compositions, medical devices, kits, or articles of manufacture.

[0146] The pH-dependent conditionally active anti-B7-H3 polypeptides, antibodies or antigenbinding antibody fragments thereof have anti-proliferative activity. Any of the anti-B7-H3 polypeptides, antibodies or antigen-binding antibody fragments thereof, multispecific antibodies and antigen-binding fragments thereof of the present application can be formulated into a pharmaceutical composition, including the multispecific antibodies and antigen-binding fragments thereof that have both pH-dependent conditional binding activity toB7-H3 and CD3 described above. Thus, the anti-B7-H3 polypeptides, antibodies, antigen binding antibody fragments thereof, or multispecific antibodies or antigen-binding antibody fragments thereof may be useful for treating proliferative diseases associated with B7-H3 protein expression. The polypeptides, antibodies, antigen binding antibody fragments thereof, or multispecific antibodies and antigen-binding fragments thereof may¬ be used alone or in combination with any suitable agent or other conventional treatments.

[0147] The anti-B7-H3 polypeptides, antibodies or antibody fragments may be used to treat diseases associated with B7-H3 protein expression, overexpression or activation. There are no particular limitations on the types of cancer or tissue that can be treated other than the requirement for B7-H3 protein expression.

[0148] In each of the aspects of the treatment methods described herein, the anti- B7-H3 polypeptide, antibody, antigen binding antibody fragment thereof, or multispecific antibody or antigen-binding antibody fragment thereof, may be delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the application herein, an effective amount of the polypeptide, antibody, antigen binding antibody fragment thereof, or multispecific antibody is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[0149] Thus, an aspect of the application relates to a method for treating a disease associated with the expression of B7-H3 protein comprising administering to a subject in need thereof with aBIAT-1039WO PATENT APPLICATIONtherapeutically effective amount of the polypeptide, antibody, antigen binding antibody fragment, or multispecific antibody or antigen-binding antibody fragment thereof, of the present application.

[0150] For administration, the anti-B7-H3 polypeptide, antibody, antigen-binding antibody fragment thereof or multi-specific antibody or antigen-binding antibody fragment thereof may be formulated as a pharmaceutical composition. The pharmaceutical composition including anti-B7-H3 antibody, antibody fragment can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution or composition containing a pharmaceutically acceptable carrier.

[0151] The pH-dependent conditionally active polypeptides, antibodies, antigen-binding antibody fragments thereof and multi-specific antibodies or antigen-binding antibody fragments thereof may be formulated as an injectable formulation. Typically, injectable compositions are prepared as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified with the pH-dependent conditionally active polypeptides, antibodies and multi-specific antibodies encapsulated in liposome vehicles.

[0152] The pH-dependent conditionally active polypeptides, antibodies, antigen-binding antibody fragments thereof and multi-specific antibodies and antigen-binding fragments thereof may be administered to a patient / subject using any available method and route suitable for drug delivery7, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the pharmaceutical composition and / or the desired effect. The treatment can be administered in a single dose or in multiple doses. In some aspects, the treatment is administered orally. In some aspects, the treatment is administered via an inhalational route. In some aspects, the treatment is administered intranasally. In some aspects, the treatment is administered locally. In some aspects, the treatment is administered intracranially. In some aspects, the treatment is administered intravenously.

[0153] A pharmaceutically acceptable carrier is a material that can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable earner. Other suitable pharmaceutically acceptable carriers are well-known to those in the art. (See, e.g. , Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)) Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.BIAT-1039WO PATENT APPLICATION

[0154] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. These considerations can be evaluated by a skilled person to formulate suitable pharmaceutical compositions. The pharmaceutical compositions of the application can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

[0155] Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. Exemplary vehicles may be isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition of, for example, sterilized water or physiological saline, permit the constitution of injectable solutions.

[0156] In some aspects, the pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form. The lyophilized preparation is typically reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add a volume of pure water (typically about equivalent to the volume removed during lyophilization). Solutions comprising antibacterial agents may also be used for the production of pharmaceutical compositions for parenteral administration; see also Chen, Drug Dev IndPharm, vol. 18, pp. 1311-54, 1994.

[0157] In some aspects, tonicity agents, sometimes known as “stabilizers'’ are present to adjust or maintain the tonicity of a liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intramolecular interactions. Tonicity agents can be present in any amount offrom0.1%to 25%by weight, for example 1 to 5% of the pharmaceutical composition. Particular tonicity agents include polyhydnc sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0158] A pharmaceutically acceptable tonicity agent may be included in the composition to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some aspects, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may also be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. TonicityBIAT-1039WO PATENT APPLICATIONagents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.

[0159] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients may include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine. 2-phenylalanine. glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0160] A pharmaceutically acceptable surfactant may be added to the composition to reduce aggregation of the formulated multi-specific antibody and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters, polyoxyethylene alkyl ethers, alkylphenylpolyoxyethylene ethers (Triton-X™), poly oxy ethylene-polyoxypropylene copolymer (Poloxamer, Pluronic™). and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij ™. Exemplary concentrations of surfactant in the composition may range from about 0.001% to about 1% w / v.

[0161] Non-ionic surfactants or detergents (also known as “wetting agents”) may be employed to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, more particularly about 0.07 mg / ml to about 0.2 mg / ml.

[0162] Suitable non-ionic surfactants include polysorbates (20, 40. 60. 65. 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethersBIAT-1039WO PATENT APPLICATION(TWEEN®-20, TWEEN®-80, etc ), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60. glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0163] A lyoprotectant may be added to the composition in order to protect the labile active ingredient (e.g. a protein) against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol and glycerol), and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 nM to 500 nM.

[0164] In some aspects, the composition, containing one or more of a surfactant, a buffer, a stabilizer, and a tonicity agent, is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other aspects, a preservative selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof, may be is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).

[0165] The doses used for the administration can be adapted as a function of various parameters, such as the mode of administration, the relevant pathology, and / or the desired duration of treatment.

[0166] To prepare pharmaceutical compositions, an effective amount of the polypeptide, antibody, antigen-binding antibody fragment thereof, multi-specific antibody or the antigen-binding antibody fragment thereof may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0167] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in a water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0168] The polypeptide, antibody, antigen-binding antibody fragment, multi-specific antibody or antigen-binding antibody fragment thereof can be formulated into a composition in a neutral or saltBIAT-1039WO PATENT APPLICATIONform. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0169] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be useful to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0170] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with one or more of the other ingredients enumerated above, as may be required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0171] The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of dimethyl sulfoxide (DMSO) as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

[0172] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0173] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous andBIAT-1039WO PATENT APPLICATIONintraperitoneal administration. In this connection, sterile aqueous media which can be employed are known to those of skill in the art. For example, a dose could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, '‘Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0174] In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently used.

[0175] In certain aspects, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0176] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafme particles (sized around 0.1 pm) are generally designed using polymers able to degrade in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present application.

[0177] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations

[0178] Pharmaceutical formulations as described herein may be prepared by mixing such antibody or antibody fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, buty l or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins.BIAT-1039WO PATENT APPLICATIONsuch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants such as polyethylene gly col (PEG).

[0179] Exemplary pharmaceutically acceptable carriers herein may include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminogly canases such as chondroitinases.

[0180] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and W02006 / 044908, the latter formulations including a histidine-acetate buffer.

[0181] The formulation herein may also contain more than one active ingredient as necessary for the indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other may be combined into a single formulation. For example, it may be desirable to provide an EGFR antagonist (such as erlotinib), an anti-angiogenic agent (such as a VEGF antagonist which may be an anti-VEGF antibody) or a chemotherapeutic agent (such as a taxoid or a platinum agent) in addition to the anti-B7-H3 polypeptide, antibody, antigen binding antibody fragment, or multispecific antibody of the present application. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0182] In one aspect, the polypeptides, antibodies, antigen-binding antibody fragments thereof, multi-specific antibodies and antigen- binding antibody fragments thereof of the present application are combined in a formulation with another antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, EpCAM, B7-H3, ROR1, SFRP4 and a WNT protein including WNT1, WNT2, WNT2B. WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A. WNT9B, WNT10A. WNT 10B. WNT11. WNT 16. The combination may be in the form of two separate molecules: the active ingredients of the present application, and the other antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule with binding affinity to both B7-H3 protein and an antigen selected from the foregoing list, thus forming a multispecific (eg. bispecific) pH-dependent conditionally active antibody.BIAT-1039WO PATENT APPLICATION

[0183] Active ingredients may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization. For example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsions may be employed. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980).

[0184] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, e.g. films, or microcapsules.

[0185] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.Therapeutic Methods and Compositions

[0186] In another aspect, the application provides a method of treatment of cancers and tumors using the anti-B7-H3, pH-dependent conditionally active polypeptides, antibodies, antigen-binding antibody fragments thereof, multi-specific antibodies and antigen-binding fragments thereof (hereinafter collectively referred to as “the actives”) described herein. The method involves administering one or more of the actives to a subject with a cancer or tumor.

[0187] In some aspects, the actives are administered in conjunction with a cancer neoantigen vaccine, or administered before or after the administration of the cancer neoantigen vaccine. Neoantigen vaccine and its generation is described in US2017 / 0202939.

[0188] Any of the actives and pharmaceutical compositions containing them provided herein may be used in therapeutic methods. In one aspect, the actives and pharmaceutical compositions containing them for use as a medicament are provided. In further aspects, the actives for use in treating cancer are provided.

[0189] Cancers that have been found to express or overexpress B7-H3 that are suitable targets for the therapeutic methods of the application include, but are not limited to Carcinoma (CA), Castration-Resistant Prostate Cancer (CRPC), Central Nervous System (CNS)ZLeptomeningeal Metastases (LM), Clear Cell Carcinoma (CCC) of the ovary, Colorectal Cancer (CRC). Epithelial Ovarian Cancer (EOC), Esophageal Squamous Cell Carcinoma (ESCC), Head And Neck Squamous Cell Carcinoma (HNSCC), Head and Neck Squamous Cell Carcinoma (HNSCC), Lentigo Malignant Melanoma (LMM), Medulloblastoma (MB), Melanoma, Metastatic Castration-Resistant Prostate Cancer (mCRPC), Muscle Invasive Bladder Cancer (MIBC), Neuroblastoma (NBL), Non-MuscleBIAT-1039WO PATENT APPLICATIONInvasive Bladder Cancer (NMIBC), Non-Small Cell Lung Cancer (NSCLC), Prostate Cancer (PCa), Renal Cell Carcinoma (RCC), Retinoblastoma (Rb). Rhabdomyosarcoma (RMS), Small Cell Lung Cancer (SCLC), Squamous Non-Small-Cell Lung Cancer (sqNSCLC), Triple-Negative Breast Cancer (TNBC), Upper Tract Urothelial Cancer (UTUC), Urothelial Carcinoma (UC or TCC), and Uveal Melanoma (UM).

[0190] In certain aspects, the actives and pharmaceutical compositions containing them are used in a method of treatment. The actives and pharmaceutical compositions containing them may be used in a method of treating an individual that has cancer comprising administering to the individual a therapeutically effective amount of one or more of the actives or the pharmaceutical compositions containing the actives.

[0191] The method may further include administering to the individual a therapeutically effective amount of at least one additional therapeutic agent. The actives and pharmaceutical compositions containing the actives may be used for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function and methods of treating these conditions. The treatment involves administering to the individual an effective amount of the at least one active sufficient to treat the condition. An “individual” according to any of the aspects of the application is preferably a human.

[0192] In a further aspect, the application provides for the use of the actives in the manufacture or preparation of a medicament. In one aspect, the medicament is for treatment of any of the cancers or diseases mentioned above. The medicament may be used in a method of treating cancer comprising administering to an individual having cancer a therapeutically effective amount of the medicament. The method may further include administering to the individual a therapeutically effective amount of at least one additional therapeutic agent as described above. In a further aspect, the medicament is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e , intratumoral vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function.

[0193] In a further aspect, the application provides a method for treating cancer. The method for treating cancer involves administering to an individual having such cancer a therapeutically effective amount of at least one of the actives or a pharmaceutical composition containing at least one of the actives. This method may also further include administering to the individual a therapeuticallyBIAT-1039WO PATENT APPLICATIONeffective amount of at least one additional therapeutic agent. In an aspect, the application provides for a method of inhibiting cell proliferation.

[0194] The additional therapeutic agent may be selected from an anti-angiogenic agent, a VEGF antagonist such as an anti-VEGF antibody, for example bevacizumab, an EGFR antagonist such as erlotinib, a chemotherapeutic agent and / or a cytostatic agent, a taxoid such as paclitaxel, a platinum agent such as carboplatinum and / or an agent that enhances the patient’s immunity or immune system.

[0195] Combination therapies encompass combined administration whereby two or more therapeutic agents are included in the same or separate formulations, and separate administration, in which case, administration of the active or pharmaceutical composition containing the active can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. The actives and pharmaceutical compositions containing them can also be used in combination with radiation therapy.

[0196] The actives may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the disorder being treated, the mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivers’ of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The active may optionally be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the active employed in the formulation, the type of disorder or treatment, and other factors discussed above.

[0197] The dosage of the active or pharmaceutical compositions containing the active will be the same if administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment.

[0198] In one aspect, the active or pharmaceutical composition containing the active may be administered together with an immune checkpoint inhibitor molecule. The combination can be the anti-B7-H3, pH-dependent conditionally active polypeptide, antibody, antigen-binding antibody fragment thereof, the multi-specific antibody or antigen-binding antibody fragment thereof and the immune checkpoint inhibitor molecule administered as a separate molecule(s) or as a multispecific antibody.

[0199] The immune checkpoint may be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, 0X40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences, vol. 20, 158, 2019). Additional immune checkpoints include B7-H4, KIR, A2aR. CD27, CD70, DR3, and ICOS (Wang et al., “Immune checkpoint blockade and itsBIAT-1039WO PATENT APPLICATIONcombination therapy with small-molecule inhibitors for cancer treatment,’' Biochim Biophys Acta Rev Cancer 1871(2): 199-224 (2019). The immune checkpoint is preferably CTLA4, PD-1 or PD-L1.

[0200] Enhancing the host's immune function to combat tumors may be used in conjunction with the methods of the present application. Conventional methods include (i) APC enhancement, such as (a) injection into the tumor of DNA encoding foreign MHC alloantigens, or (b) transfecting biopsied tumor cells with genes that increase the probability of immune antigen recognition (e ., immune stimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2) of the tumor, (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T-cells. Adoptive cellular immunotherapy includes isolating tumor-infiltrating host T-lymphocytes, expanding the population in vitro, such as through stimulation by IL-2 or tumor or both. Additionally, isolated T-cells that are dysfunctional may also be activated by in vitro application of anti-PD-Ll antibodies. T-cells that are so-activated may then be readministered to the host. One or more of these methods may be used in combination with the treatments of the present application described above.

[0201] Traditional therapies for cancer include the following: (i) radiation therapy (e.g, radiotherapy, X-ray therapy, irradiation) or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered either externally via external beam radiotherapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, or the application of cytotoxic drug which generally affect rapidly dividing cells; (iii) targeted therapies, or agents which specifically affect the deregulated proteins of cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancement of the host's immune response (e.g, vaccine); (v) hormonal therapy, or blockade of hormone (e.g, when tumor is hormone sensitive), (vi) angiogenesis inhibitor, or blockade of blood vessel formation and growth, and (vii) palliative care, or treatment directed to improving the quality7of care to reduce pain, nausea, vomiting, diarrhea and hemorrhage. Pain medication such as morphine and oxycodone, anti-emetics such as ondansetron and aprepitant, can permit more aggressive treatment regimens.

[0202] In the treatment of cancer, any of these conventional treatments for cancer or enhancing immunity may be conducted, prior to, subsequent to or simultaneous with the administration of the actives or pharmaceutical compositions containing the actives.Articles of Manufacture and Kits

[0203] The actives of the present application may be included in pharmaceutical compositions, and the actives and pharmaceutical compositions may be included in medical devices, kits, and articles of manufacture for therapeutic or prophylactic use. Suitable pharmaceutical compositions, medical devices, kits, or articles of manufacture are described in detail in WO 2016 / 138071.BIAT-1039WO PATENT APPLICATION

[0204] The article of manufacture may include a container and a label or package insert on or associated wi th the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating and / or preventing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0205] At least one active agent in the composition is an active or pharmaceutical composition containing the active of the present application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody or antibody fragment; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherw ise therapeutic agent. The article of manufacture in this aspect of the application may further comprise a package insert indicating that the compositions can be used to treat a condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically -acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0206] Finally, the application also provides kits including at least one active or pharmaceutical composition containing the active of the present application and one or more of an additional therapeutic agent or an article of manufacture for administration or dosing of the active or pharmaceutical composition. The kits further contain instructions on the use thereof.

[0207] The following examples are illustrative, but not limiting, of the methods of the present application. Other suitable modifications and adaptations of the variety7of conditions and parameters normally encountered in the field, and which are obvious to those skilled in the art, are within the scope of the application.BIAT-1039WO PATENT APPLICATIONEXAMPLESExample 1: Enzyme-linked Immunosorbent Assays (ELISA) protocol1. One day before ELISA, a 96 well plate was coated with 100 pl of 0.5 pg / ml recombinant protein (e.g., antigen) overnight in ELISA coating buffer at 4 °C.2. Dilute antibody samples in ELISA assay buffer.3. Flicked off buffer from the plate coated with antigen, blot dry on paper towels.4. Block plate with 200 pl ELISA assay buffer at room temperature for 1 hour.5. Add 100 pl of diluted samples to each well.6. Incubate the plate at room temperature for 1 hour.7. Prepare the secondary antibody in screening buffers according to the layout of the plate.8. Flicked off buffer from the plate, blot dry’ on paper towels.9. Wash the plate for a total of 3 times with ELISA wash buffer.10. Add 100 pl of diluted corresponding secondary7antibodies (anti -human HRP secondary antibody , anti-His antibody and anti-mouse HRP secondary' antibody) in ELISA assay buffer to the wells:12. Incubate the plate at room temperature for 1 hour.13. Flicked off buffer from the plate, blot dry on paper towels.14. Wash the plate for a total of 3 times with ELISA wash buffer.15. Flick off buffers from plate, blot dry on paper towels.16. Add 50 pl of 3,3',5,5’-Tetramethylbenzidine (TMB) substrate according to the plate layout.17. Stop development with 50 pl 0.1N HC1.18. Read at OD450 nm using a plate readerExample 2: pH Affinity ELISA

[0208] The human B7-H3-his recombinant protein was immobilized in the w ells of a 96-w ell plate overnight at 4°C. Plates were blocked with either pH 6.0 ELISA assay solution (PBS with 2.5g / L sodium bicarbonate and 1% BSA) or pH7.4 ELISA assay solution at room temperature for one hour, and then washed with the corresponding pH ELISA assay solution. The WT-B7-H3 CD3, the DualCAB-CPSl 1 x CD3-BF45 and the DualCAB-CPSl 1 x CD3-BF46 antibodies were serially diluted in the corresponding pH ELISA assay solutions and added to the wells. ELISA plates containing diluted antibodies were sealed and incubated at room temperature for one hour with shaking. The plates were then washed three times with the corresponding pH ELISA assay solution.BIAT-1039WO PATENT APPLICATIONAnti-human IgG antibody HRP secondary antibody was added to each well. The plates were then sealed and incubated at room temperature for one hour with shaking. Following incubation, the plates were washed thrice with the corresponding pH ELISA assay solution. TMB peroxidase substrate solution was added to each well and the reaction was stopped with 0. IN HC1. The OD at 450 nm was measured using a Microplate Spectrophotometer. EC50 values for binding to human B7-H3 at pH 6.0 and pH 7.4 were determined using the nonlinear fit model (variable slope, four parameters) of GraphPad Prism version 7.03.

[0209] The binding curves are presented in Figures 3A and 3B. The EC50 values at pH 6.0, at pH 7.4, and the ratio of binding are presented in Table 4 below.Table 4: ELISA EC50 values at pH 6.0, at pH 7.4Example 5: pH Sandwich Affinity ELISACD3 Capture and huB7-H3-mFc detection or cyno B7-H3 ECD

[0210] The human CD3e / 5 heterodimer was immobilized in the wells of a 96-well plate overnight at 4°C. Plates were blocked with either pH 6.0 ELISA assay solution (PBS with 2.5g / L sodium bicarbonate and 1% BSA) or pH 7.4 ELISA assay solution at room temperature for one hour, and then washed with the corresponding pH ELISA assay solution. The WT-B7-H3 CD3, the DualCAB-CPS11 x CD3-BF45 and the DualCAB-CPSll x CD3-BF46 antibodies were serially diluted in the corresponding pH ELISA assay solutions and added to the wells. ELISA plates containing diluted antibodies were sealed and incubated at room temperature for one hour with shaking. The plates were then washed three times with the corresponding pH ELISA assay solution. Next, human or cyno B7-H3 ECD fused to mouse Fc protein and diluted in the corresponding pH ELISA assay solution was added to each well. The plates were sealed and incubated at room temperature for one hour with constant shaking. Following incubation, the plates were washed three times with the corresponding pH ELISA assay solution, and anti-mouse IgG antibody HRP secondary antibody diluted in the corresponding pH ELISA solution was added to each well. The plates were then sealed and incubated at room temperature for one hour with shaking. Following incubation, the plates were washed twice with the corresponding pH ELISA assay solution. TMB peroxidase substrate solution was added toBIAT-1039WO PATENT APPLICATIONeach well and the reaction was stopped with 0.1N HC1. The OD at 450 nm was measured using a Microplate Spectrophotometer. EC50 values for binding to huCD3 / huB7-H3-mFc and huCD3 / cynoB7-H3-mFc at pH 6.0 and pH 7.4 were determined using the nonlinear fit model (variable slope, four parameters) of GraphPad Prism version 7.03.

[0211] The binding curves for human CD3 capture and huB7-H3-mFc detection are presented in Figures 4A-4B. The EC50 values at pH 6.0 and at pH 7.4, and the ratio of binding at pH 6.0 / pH 7.4 are presented in Table 5 below.Table 5: EC 50 values: HumCD3 capture, humB7-H3-mFc / anti-mouse IgG Detection

[0212] The EC50 values at pH 6.0 and at pH 7.4, and the ratio of binding at pH 6.0 / pH 7.4 are presented in Table 6 below.Table 6: EC50 values HumCD3 capture, cynoB7-H3-mFc / anti-mouse IgG DetectionExample 6: Specificity of DualCAB-CPSll x CD3-BF45 binding

[0213] The human B7-H3-his recombinant protein was immobilized in the wells of a 96-well plate overnight at 4°C. Plates were blocked with either pH 6.0 ELISA assay solution (PBS with 2.5 g / L sodium bicarbonate and 1% BSA) or pH 7.4 ELISA assay solution at room temperature for one hour, and then washed with the corresponding pH ELISA assay solution. The WT-B7-H3 CD3, the DualCAB-CPSll x CD3-BF45 and the related antibodies anti-PD-Ll; anti-PDlL2; anti-B7-2, and anti-B7-H2 were diluted in the corresponding pH ELISA assay solutions and added to the wells. ELISA plates containing diluted antibodies were sealed and incubated at room temperature for one hour with shaking. The plates were then washed three times with the corresponding pH ELISA assay solution. Anti-human IgG antibody HRP secondary antibody was added to each well. The plates were then sealed and incubated at room temperature for one hour with shaking. Following incubation, theBIAT-1039WO PATENT APPLICATIONplates were washed thrice with the corresponding pH ELISA assay solution. TMB peroxidase substrate solution was added to each well and the reaction was stopped with 0. IN HC1. The OD at 450 nm was measured using a Microplate Spectrophotometer. Binding signals to human B7-H3 at pH 6.0 and pH 7.4 were plotted with GraphPad Prism version 7.03.

[0214] The absorbance values at 450 nm are presented in Figure 6.Example 7: pH Cross Species ELISA; human, cyno, rat, and mouse antigens

[0215] The human, cyno, rat and mouse B7-H3-his recombinant proteins were immobilized in the wells of a 96-well plate overnight at 4°C. Plates were blocked with either pH 6.0 ELISA assay solution (PBS with 2.5 g / L sodium bicarbonate and 1% BSA) or pH 7.4 ELISA assay solution at room temperature for one hour, and then washed with the corresponding pH ELISA assay solution. The WT-B7-H3 CD3 and the DualCAB-CPSll x CD3-BF45 antibodies were diluted in the corresponding pH ELISA assay solutions and added to the wells. ELISA plates containing diluted antibodies were sealed and incubated at room temperature for one hour with shaking. The plates were then washed three times with the corresponding pH ELISA assay solution. Anti-human IgG antibody HRP secondary antibody was added to each well. The plates were then sealed and incubated at room temperature for one hour with shaking. Following incubation, the plates were washed thrice with the corresponding pH ELISA assay solution. TMB peroxidase substrate solution was added to each well and the reaction was stopped with 0.1N HC1. The OD at 450 nm was measured using a Microplate Spectrophotometer. Binding signals to human, cyno, rat and mouse B7-H3 at pH 6.0 and pH 7.4 were plotted with GraphPad Prism version 7.03.

[0216] The absorbance values at 450 nm are presented in Figure 7.Example 8: B7-H3 / CD3 bispecific antibody T-Cell activation assay with A375 Cells and Detroit 562 cells

[0217] A T-cell Activation Bioassay (Promega, catalog no. J1621) was used to evaluate the functional activity of DualCAB CPS 11 BF45 and DualCAB CPS 11 BF46 antibodies. Target cell lines, A375 (American Type Culture Collection, ATCC) and Detroit562 (ATCC), both expressing human B7-H3 were seeded at a density of 4x104cells / well in culture media (DMEM plus 10% FBS) overnight at 37°C with 5% CO2. The next day, the DualCAB CPS 11 BF45 and the DualCAB CPS 11 BF46 antibodies were serially diluted in DMEM 10% FBS at pH 6.0 and pH 7.4. Engineered Jurkat TCR / CD3 cells that express a luciferase reporter driven by an NFAT-response element were thawed and resuspended to 4xl06cells / mL in DMEM 10% FBS at pH 6.0 and pH 7.4. Media of the cells was removed: 25 pL of diluted antibodies were added at each respective pH value to the cells, and 25 pL of TCR / CD3 effector cell suspension at pH 6.0 and pH 7.4 was added to the same wells at eachBIAT-1039WO PATENT APPLICATIONrespective pH value. Co-cultures of target and effector cells were incubated with the antibodies at 37°C 5% CO2 for 6 hours. After this period, 50 pL / well of Bio-Gio reagent was added to each well and the cultures were incubated for 5 minutes at room temperature. The luminescence signal was quantified using a SpectraMax-i3X plate reader (Molecular Devices). The data was analyzed using GraphPad Prism Software 7.03.

[0218] The EC50 (ug / mL) in A375 cells and Detroit 562 cells are presented in Table 7.Table 7: T cell Activation Assay -A375 and Detroit 562 Cells• N.D.= not determined. A complete saturated curve was not obtained. EC50 values are not accurate• N / A = not applicableExample 9: pH FACS of Jurkat, A375, and Detroit562 cells

[0219] The binding affinity of the DualCAB CPS 11 BF45 and DualCAB CPS 11 BF46 antibodies to B7-H3 and CD3 expressing cells was evaluated by flow cytometry. The binding activity of these antibodies to human B7-H3 was evaluated using A375 (ATCC) and Detroit562 (ATCC) cancer cells. The binding activity of these antibodies to human CD3 was evaluated using Jurkat cells (ATCC).3x105cells were incubated for 1 hour on ice with 100 pL of each of the antibodies at 10, 1 and O.lpg / mL in PBS 1% BSA 2.5 g / L of sodium bicarbonate at pH 6.0 or at pH 7.4. After this period, cells were washed two times with each respective buffer. Binding of the DualCAB antibodies was detected by resuspending the cells with 100 pL secondary anti-hlgG AF488 conjugated antibody diluted 1:200 in PBS 1% BSA 2.5 g / L of sodium bicarbonate buffer at pH 6.0 or at pH 7.4. Cells were incubated again for 45 minutes on ice and washed three times with each respective buffer. After the last wash, the cells were fixed with PBS 1% paraformaldehyde for 10 minutes, in the dark at room temperature, washed one more time in PBS and then analyzed by fluorescence-activated cell sorting (FACS) using aNovocyte Flow Cytometer and software. For calculations of EC50 values, a similar protocol was used to generate a curve-dose response, the antibodies were serially diluted 1:3 from 100 pg / mL to 0.046 pg / mL.

[0220] The mean fluorescence intensities (MFI) in Jurkat cells are presented in Figures 8A-8B.

[0221] The mean fluorescence intensities (MFI) in A375 cells are presented in Figures 9A-9B.BIAT-1039WO PATENT APPLICATION

[0222] The mean fluorescence intensities (MFI) in Detroit562 cells are presented in Figures 10A-10B.

[0223] The mean fluorescence intensities (MFI) in A375 cells in an embodiment is presented in Figures 11 A-l IB. The EC50 values at pH 6.0 and at pH 7.4 are presented in Table 8 below.Table 8: EC50 values at pH 6.0 and pH 7.4 in A375 cellsThe mean fluorescence intensities (MFI) in Detroit 562 cells are presented in Figures 12A-12B.Example 10: Effects of DualCAB CPS 11 BF45 on humanized mouse tumor model DualCAB A375 CDX humanized mouse model

[0224] Female NCG mice (NOD Pkrdc, IL2r gamma triple-immunodeficient established by GRISPR / Cas9 technology) were inoculated subcutaneously into the right lower flank of mice with 0.1 mL of A375 cells inMatrigel (1:1). The total number of cells implanted was 5 x 106 / mouse. After tumor inoculation the mice were engrafted with 5xl06human peripheral blood mononuclear cells (PBMCs) inoculated intravenously (IV). Animals were randomized to treatment groups when tumor volume reached approximately 80-100 mm3. Following randomization, animals were dosed intravenously with isotype x CAB CD3 BF45 control antibody or DualCAB CPS 11 BF45 antibody at 2 mg / kg twice a week for four weeks.

[0225] A graph of the mean tumor volume versus time of is shown in Figure 13.Detroit562 CDX humanized mouse model

[0226] Female NCG mice (NOD Pkrdc, IL2r gamma triple-immunodeficient established by GRISPR / Cas9 technology) were inoculated intraperitoneally with 5xl06human PBMCs on day 1. Detroit562 human pharyngeal carcinoma was implanted subcutaneously in the right lower flank of the mice on day 0 (10xl06cells / mouse). Animals were randomized to treatment groups when tumor volume reached approximately 120 mm3. Following randomization, animals were dosed intravenously with isoty pe x CAB CD3 BF45 control antibody at 2 mg / kg or with DualCAB CPS 11 BF45 at 2. 1 and 0.5 mg / kg twice a week for four weeks.

[0227] A graph of the mean tumor volume versus time is shown in Figure 14.BIAT-1039WO PATENT APPLICATIONExample 11: Non-GLP Toxicity in Non-human primates (NHP)

[0228] The toxicity and toxicokinetics of the DualCAB CPS 11 BF45 antibody was evaluated after a single or repeat dose at escalating dose levels via intravenous (bolus) injection in cynomolgus monkeys. Male and female monkeys (1 / gender / group) were dosed with DualCAB CPS 11 BF45 antibody at 5 and 25 mg / kg once, or at 1, 3, 10 and 25 mg / kg once weekly for up to three weeks. Assessment of toxicity was based on mortality7, clinical observations, body weights, food consumption, and clinical and anatomic pathology. Blood samples were collected for toxicokinetic evaluations, peripheral blood immunophenotyping. and cytokine analysis.

[0229] No DualCAB CPS 11 BF45-related effects on clinical observations, body weights, food consumption, clinical pathology parameters, peripheral blood immunophenotyping, or anatomic pathology findings were noted for animals administered single or repeat doses of up to 25 mg / kg. DualCAB CPS 11 BF45-related changes in cytokines were limited to transient increases in MCP-1; transient decreases in IL-8 and sporadic increases in IL-6 that were noted with an uncertain relationship to DualCAB CPS 11 BF45. Due to the mild severity of the findings and the lack of an impact on the health and well-being of the animals that received the DualCAB CPS 11 BF45-related antibody doses of up 25 mg / kg / dose once or once weekly for up to three weeks, the maximum tolerated dose (MTD) for DualCAB CPS 11 BF45-related antibody was determined to be 25 mg / kg / dose after a single or repeat dosing. There was no significant accumulation in TK exposure after three w eekly intravenous administrations, consistent with the observed Tl / 2 ranging from 12.0 to 26.3 hours after a single intravenous administration and 12.0 to 31.2 hours after a third weekly intravenous administration.

[0230] All documents mentioned herein are hereby incorporated by reference in their entirety and at least to provide the disclosure for which they were specifically relied upon or cited as referring to. The applicant does not intend to dedicate any disclosed aspects to the public, and to the extent any disclosed modifications or alterations may not literally fall within the scope of the claims, they are considered to be part hereof under the doctrine of equivalents.

[0231] It is to be understood, however, that even though numerous characteristics and advantages of the present application have been set forth in the foregoing description, together with details of the structure and function of the invention, the disclosure is illustrative only, and changes may be made in detail, especially in maters of shape, size and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meanings of the terms in which the appended claims are expressed.BIAT-1039WO PATENT APPLICATIONSEQUENCESSEQ ID NO:1 hCDRl B7-H3 GFDFSSFGEHSEQ ID NO: 2 hCDR2 B7-H3 YISSDSSAIYYADTVKGSEQ ID NO: 3 hCDR3 B7-H3 GRENIYYGARLDYSEQ ID NO:4 1CDR1 B7-H3 KASQNVDDQVASEQ ID NO: 5 1CDR2 B7-H3 SASYRYSSEQ ID NO: 6 1CDR3 B7-H3 QQYNKYPFTSEQ ID NOY hCDRl CD3 GFTFNTYAMNSEQ ID NO: 8 hCDR2 CD3 RIRSKYNNYATYYADSVKDSEQ ID NO: 9 hCDR3 CD3a HSNFGNSKVSWFAYSEQ ID NOTO hCDR3 CD3b HTNFGNSKVSWFAYSEQ ID NO: 11 1CDR1 CD3 RS S AGAVTTSNYDNSEQ ID NO: 12 1CDR2 CD3 GTNKRAPSEQ ID NO: 13 1CDR3 CD3 ALWYSNLWVSEQ ID NO: 14 CPS1I-LC EIVMTQSPATLSVSPGERATLSCKASQNVDDQVAWFQQRPGQSPRRLIYSAS YRYSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNKYPFTFGQGTKVEIK SEQ ID NO: 15 CPS11-HC QVQLVQSGAEVKKPGASVKVSCKASGFDFSSFGEHWIRQPPGKGLEWIGYISSDSS AIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCGRGRENIYYGARLDY WGQGTLVTVSS SEQ ID NO: 16 CPS11-BF45-LC EIVMTQSPATLSVSPGERATLSCKASQNVDDQVAWFQQRPGQSPRRLIYSASYRYS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNKYPFTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQL VESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHSNFGNSKVSWFA YWGQGTLVTVSSGGSGGSGGSGGSGGQAVVTQEPSLTVSPGGTVTLTCRSSAGAV TTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAED EADYYCALWYSNLWVFGGGTKLTVLSR SEQ ID NO: 17 CPS11-BF46-LCEIVMTQSPATLSVSPGERATLSCKASQNVDDQVAWFQQRPGQSPRRLIYSASYRYSBIAT-1039WO PATENT APPLICATIONGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNKYPFTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQL VESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHTNFGNSKVSWFA YWGQGTLVTVSSGGSGGSGGSGGSGGQAVVTQEPSLTVSPGGTVTLTCRSSAGAV TTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAED EADYYCALWYSNLWVFGGGTKLTVLSR SEQ ID NOT8 CPS11-BF45-HC QVQLVQSGAEVKKPGASVKVSCKASGFDFSSFGEHWIRQPPGKGLEWIGYISSDSS AIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCGRGRENIYYGARLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 19 Anti-CD3 scFv-1 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSK YNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHSNFGNSKV SWFAYWGQGTLVTVSSGGSGGSGGSGGSGGQAVVTQEPSLTVSPGGTVTLTCRSS AGAVTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGA Q AEDEADYYC ALWYSNLWVFGGGTKLTVL SR SEQ ID NO:20 Anti-CD3 scFv-2EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA RIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHTNF GNSKVSWFAYWGQGTLVTVSSGGSGGSGGSGGSGGQAVVTQEPSLTVSPGGTVTL TCRSSAGAVTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAAL TITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR SEQ ID NO:21 HGNFGNSYVSWFAYSEQ ID NO 22 HSNFGNSKVSWFAYSEQ ID NO 23 HGNFPNSKVSWFQYSEQ ID NO:24 HSNFGNSKVSWFAYBIAT-1039WO PATENT APPLICATIONSEQ ID NO 25 RSSTGAVTTSNYANSEQ ID NO 26 RSSTGAVTTSNYDN62

Claims

BIAT-1039WO PATENT APPLICATIONWHAT IS CLAIMED IS:

1. An isolated polypeptide having a pH-dependent conditionally active antigen binding activity to a B7-H3 (CD276) antigen, the polypeptide comprising:a heavy chain variable region comprising three complementarity determining regions (CDRs) having amino acid sequences Hl, H2, and H3, wherein:the Hl sequence is GFDFSSFGEH (SEQ ID NO: 1);the H2 sequence is YISSDSSAIYYADTVKG (SEQ ID NO:2); and the H3 sequence is GRENIYYGARLDY (SEQ ID NO:3);a light chain variable region including three complementarity determining regions having amino acid sequences LI, L2, and L3, wherein:the LI sequence is KASQNVDDQVA (SEQ ID NO:4);the L2 sequence is SASYRYS (SEQ ID NO:5); andthe L3 sequence is QQYNKYPFT (SEQ ID NO:6);wherein the isolated polypeptide has a higher binding activity to the B7-H3 (CD276) antigen at pH 6.0 than at pH 7.4.

2. The isolated polypeptide of claim 1, wherein said pH-dependent conditionally active antigen binding activity7to B7-H3 (CD276) comprises conditional binding to both human and cynomolgus monkey B7-H3 (CD276).

3. The isolated polypeptide of claim 1 or 2, wherein said isolated polypeptide has a ratio of B7-H3 (CD276) antigen binding activity at pH 6.0 to B7-H3 (CD276) antigen binding activity at pH 7.4 of at least 1.3 to 1.

4. The isolated polypeptide of claim 3, wherein the ratio of the B7-H3 (CD276) antigen binding activity at pH 6.0 to the B7-H3 (CD276) antigen binding activity at pH 7.4 is from 2 to 20.

5. The isolated polypeptide of any one of claims 1 to 4, further comprising a pH-dependent conditionally active antigen binding activity to CD3 antigen, said light chain variable region including six anti-CD3 complementarity determining regions having sequences L4, L5, L6, L7, L8, and L9, wherein:the L4 sequence is GFTFNTYAMN (SEQ ID NO: 7).the L5 sequence is RIRSKYNNYATYYADSVKD (SEQ ID NO: 8),the L6 sequence is HSNFGNSKVSWFAY (SEQ ID NO: 9) or HTNFGNSKVSWFAY (SEQ ID NOTO,the L7 sequence is RSSAGAVTTSNYDN (SEQ ID NO: 11),54BIAT-1039WO PATENT APPLICATIONthe L8 sequence is GTNKRAP (SEQ ID NO: 12), andthe L9 sequence is ALWYSNLWV (SEQ ID NO: 13); andwherein said light chain variable region including the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8, and L9 binds to CD3 antigen and the isolated polypeptide has a higher binding activity' to the CD3 antigen at pH 6.0 than at pH 7.4.

6. The isolated polypeptide of claim 5, wherein said isolated polypeptide has a ratio of CD3 antigen binding activity at pH 6.0 to CD3 antigen binding activity at pH 7.4 of at least 1.3 to 1.

7. The isolated polypeptide of claim 6, wherein the ratio of the CD3 antigen binding activity at pH 6.0 to the CD3 binding activity at pH 7.4 is from 2 to 20.

8. The isolated polypeptide of any one of claims 5 to 7, wherein said light chain variable region comprising the six anti-CD3 complementarity determining regions having the sequences L4, L5, L6, L7, L8 and L9 is selected from a single chain variable fragment (scFv), a nanobody (Camelid VHH), a DARPIN, a minibody, or a tri-scFv, each having conditional CD3 antigen binding activity.

9. The isolated polypeptide of claim 8, wherein said light chain variable region comprising the six anti-CD3 complementarity' determining regions having the sequences L4, L5, L6, L7, L8 and L9 is an scFv and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical, to an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO:20.

10. The isolated polypeptide of any one of claims 1 to 9, wherein the light chain variable region that includes the CDRs having the sequences LI. L2 and L3 comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17.

11. The isolated polypeptide of any one of claims 1 to 10, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:15 and SEQ ID NO: 18.

12. The isolated polypeptide of any one of claims 1 to 11, wherein the light chain variable region that includes the CDRs having the sequences LI, L2 and L3, comprises an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NO: 17.55BIAT-1039WO PATENT APPLICATION13. The isolated polypeptide of any one of claims 1 to 12, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 18.

14. The isolated polypeptide of any one of claims 6 to 13, wherein variations from the amino acid sequences SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 18, are conservative amino acid substitutions.

15. The isolated polypeptide of any one of claims 1 to 14, wherein the binding to the B7-H3 antigen at pH 6.0 is reversible.

16. The isolated polypeptide of any one of claims 5 to 15, wherein the binding to the CD3 antigen at pH 6.0 is reversible.

17. The isolated polypeptide of any one of claims 1 to 16, wherein the isolated polypeptide is an antibody, or antigen binding antibody fragment.

18. The isolated polypeptide of claim 17, wherein the antibody or antigen binding antibody fragment is a chimeric antibody, a multispecific antibody, or a humanized antibody.

19. A multi -specific antibody comprising the isolated polypeptide of any one of claims 5 to 17.

20. The multi-specific antibody of claim 19, wherein said antibody is bispecific.

21. A method of treating a cancer in a subject in need thereof comprising administering an isolated polypeptide of any one of claims 1 to 18, or a multi-specific antibody of claim 19.

22. The method of treating a cancer of claim 21, wherein said cancer expresses a B7-H3 (CD276) antigen.

23. The method of claim 22, wherein expression of the B7-H3 (CD276) antigen is on a surface of a cancer cell.

24. The method of treating a cancer in a subject in need thereof of any one of claims claim 21 to 23, wherein said cancer is a carcinoma, adenocarcinoma, glioma, a lymphoma, a melanoma, or a cystadenoma.

25. The method of treating a cancer in a subject in need thereof of any one of claims claim 21 to 24, wherein said cancer is selected from B-cell lymphomas (Hodgkin's lymphomas and / or nonHodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular56BIAT-1039WO PATENT APPLICATIONcancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinaiy tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.

26. The method of treating a cancer in a subject in need thereof of any one of claims 21 to 24. wherein said cancer is selected from T cervical squamous cell carcinoma and endocervical adenocarcinoma, uterine corpus endometrial carcinoma, head and neck squamous cell carcinoma, liver hepatocellular carcinoma, thyroid carcinoma, bladder urothelial carcinoma, skin cuteneous melanoma, ovary serous cystadenocarcinoma, kidney chromophobe, testicular germ cell tumor, colon adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, or prostate adenocarcinoma, stomach adenocarcinoma.

27. The method of treating a cancer in a subject in need thereof of any one of claims 21 to 26, wherein the isolated polypeptide, the antibody or antigen binding antibody fragment, or the multi-specific antibody, is administered in conjunction with a cancer neoantigen vaccine.

28. The method of treating a cancer in a subject in need thereof of any one of claims 21 to 27, wherein the isolated polypeptide, the antibody or antigen binding antibody fragment, or the multi-specific antibody, is administered after administration of a cancer neoantigen vaccine.

29. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1 to 18, the multi-specific antibody of any one of claims 19 to 20, and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein said composition is a liquid form, a lyophilized form, or a liquid form reconstituted from a lyophilized form.

31. The pharmaceutical composition of claim 29 or 30, wherein said composition further comprises a tonicity agent.

32. The pharmaceutical composition of any one of claims 29 to 31, further comprising an immune checkpoint inhibitor.

33. The pharmaceutical composition of claim 32. wherein said immune checkpoint inhibitor is selected from CTLA4, LAG3, TIM3. TIGIT. VISTA, BTLA, 0X40. CD40. 4-1BB. PD-1, PD- Ll, GITR, B7-H4, KIR, A2aR, CD27, CD70, DR3, or ICOS.57BIAT-1039WO PATENT APPLICATION34. A kit for treatment, said kit comprising the isolated polypeptide of any one of claims 1 to 18, the multi-specific antibody of any one of claims 19 to 20, or the pharmaceutical composition of any one of claims 29 to 33, and instructions for using the isolated polypeptide, the antibody or antigen binding antibody fragment, the multi-specific antibody, or the pharmaceutical composition for treatment.

35. The use of the isolated polypeptide of any one of claims 1 to 18, the multi-specific antibody of any one of claims 19 to 20, or the pharmaceutical composition of any one of claims 29 to 33, for the manufacture of a medicament.

36. The isolated polypeptide of any one of claims 1 to 18, the multi-specific antibody of any one of claims 19 to, or the pharmaceutical composition of any one of claims 29 to 33, for use as a medicament.

37. The isolated polypeptide of any one of claims 1 to 18, the multi-specific antibody of any one of claims 19 to 20, or the pharmaceutical composition of any one of claims 29 to 33, for use in the treatment of cancer.

38. Use of the isolated polypeptide of any one of claims 1 to 18, , the multi-specific antibody of any¬ one of claims 19 to 20, or the pharmaceutical composition of any one of claims 29 to 33, for the manufacturing of a medicament for the treatment of cancer.

Images