Intercellular adhesion molecule 1 (ICAM1) antibody drug conjugates and their uses

Abstract

The present disclosure provides compositions comprising intercellular adhesion molecule 1 (ICAM1) antibodies and methods for using the same for therapeutic applications, such as treating triple-negative breast cancer (TNBC) and predicting drug response.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 152,747, filed February 23, 2021, entitled "Intercellular Adhesion Molecule 1 (ICAM1) Antibody Drug Conjugates and Uses Thereof," the entire contents of which are incorporated herein by reference.

[0002] Reference to sequence listing submitted as a text file via EFS-WEB This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on February 23, 2022, is named C123370193WO00-SEQ-ZJG and is 9,612 bytes in size. [Background technology]

[0003] background Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). Representing 15-20% of all breast cancers, TNBC occurs more frequently in women younger than 50 years, African American women, and in individuals with breast cancer early onset 1 (BRCA1) gene mutations. Due to a lack of therapeutic targets and limited treatment options, the prognosis of TNBC patients remains the poorest of all breast cancer patients. Summary of the Invention

[0004] overview The present disclosure is based, at least in part, on the surprising finding that targeting intercellular adhesion molecule 1 (ICAM1) can improve triple-negative breast cancer (TNBC) treatment and stratify patient populations for precision medicine. Triple-negative breast cancers grow independently of signaling mediated by several receptors typically found on the cell surface of other breast cancers, namely estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2), severely limiting the range of therapeutic options available for the treatment of these types of cancer. Due to these limitations, TNBC is characterized by the poorest prognosis of any type of breast cancer. These limitations are addressed, at least in part, by the present disclosure.

[0005] Provided herein are, in some aspects, antibody-drug conjugates (ADCs) comprising antibodies against intercellular adhesion molecule 1 (ICAM1), useful for the treatment of TNBC. As described below, the use of ADCs comprising ICAM1 antibodies allows for preferential targeting of TNBC cells over non-cancerous cells, which may improve the therapeutic window of drugs and limit toxicity. ICAM1 ADCs also exhibit improved specificity for TNBC cells than currently approved ADC therapies. Given the high genetic heterogeneity of breast cancer, predicting treatment sensitivity and responsiveness within a patient population is also challenging. Consequently, a further aspect of the present disclosure provides a method for identifying a patient population for treatment with ICAM1 antibodies or ADCs comprising ICAM1 antibodies in TNBC subjects.

[0006] An aspect of the disclosure provides a method of treating TNBC comprising administering to a subject in need thereof an effective amount of an antibody-drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to a drug.

[0007] In some embodiments, the drug is selected from the group consisting of: N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)mertansine (DM1), N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF). In some embodiments, the drug is MMAE. In some embodiments, the drug is MMAF. In some embodiments, the ICAM1 antibody and the drug are conjugated via a linker.

[0008] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is selected from the group consisting of N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), Sulfo-SPDB, valine-citrulline (Val-cit), acetylbutyrate, CL2A, and maleimidocaproyl (MC), and Mal-EBE-Mal. In some embodiments, the cleavable linker is MC. In some embodiments, the cleavable linker is Mal-EBE-Mal. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is selected from the group consisting of N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), and MC-VC-PAB. In some embodiments, the non-cleavable linker is MC-VC-PAB.

[0009] In some embodiments, the ICAM1 antibody is selected from the group consisting of an IgG, an Ig monomer, a Fab fragment, an F(ab')2 fragment, an Fd fragment, an scFv, a scAb, a dAb, an Fv, an affibody, a bispecific antibody, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the ICAM1 antibody is R6.5 or HCD54.

[0010] In some embodiments, the ICAM1 antibody is a chimeric antibody. In some embodiments, the ICAM1 antibody is a humanized antibody. In some embodiments, the ICAM1 antibody is a chimeric or humanized version of R6.5 or HCD54. In some embodiments, the ratio of ICAM1 antibody to drug in the ADC is 1: 1 to 1: 10. In some embodiments, the ratio of ICAM1 antibody to drug in the ADC is 1: 4.

[0011] In some embodiments, the ADC is administered via injection. In some embodiments, the injection is an intravenous injection. In some embodiments, the injection is an intratumoral injection. In some embodiments, the ADC is administered at a dosage of 1 mg / kg to 75 mg / kg. In some embodiments, the ADC is administered at a dosage of 5 mg / kg. In some embodiments, the ADC is administered at a frequency of once a week to once every other month.

[0012] In some embodiments, the subject to which an effective amount of the ADC is administered is a human subject. In some embodiments, the type of ICAM1-expressing cancer for which an effective amount of the ADC is administered to a subject in need thereof is breast cancer, prostate cancer, ovarian cancer, melanoma, or lung cancer. In some embodiments, the cancer is TNBC.

[0013] A further aspect of the present disclosure provides a method of treating TNBC by administering to a subject in need thereof an antibody-drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to monomethylauristatin E (MMAE) via an MC-VC-PAB linker.

[0014] A further aspect of the present disclosure provides a method of treating TNBC by administering to a subject in need thereof an antibody drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to monomethylauristatin F (MMAF) via an MC linker. [Brief description of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by a like numeral. For purposes of clarity, every component may not be shown in every drawing. In the drawings:

[0016] [Figure 1A] Figures 1A-1E show differential expression of ICAM1 in human TNBC cells versus normal cells. Figure 1A: ICAM1 mRNA levels were quantitatively compared in different breast cancer subtypes, molecular subtypes of TNBC, cancer grades of TNBC, and breast tumors with BRCA1 / 2 or TP53 mutations. ***p<0.001. [Figure 1B] FIG 1B: Human TNBC cell surface expression of ICAM1 and TROP2 was compared to normal MCF10A cells. Non-targeting IgG was used as a control. [Figure 1C-E] Figure 1C: IF staining of ICAM1 and TROP2 on human TNBC cells and normal MCF10A cells. Figure 1D: Representative imaged flow cytometry images showing cellular internalization of ICAM1 antibody in human TNBC cells and normal MCF10A cells. Figure 1E: Signal intensity analysis of ICAM1 antibody-mediated cellular internalization (n=5,000 cells).

[0017] [Figure 2A-B]Figures 2A-2C show the selective elimination of human TNBC cells by ICAM1 ADCs. Figure 2A: Schematic representation of ICAM1 ADCs. Figure 2B: DAR characterization of the four constructed ICAM1 ADCs, including IC1-MMAE, IC1-MMAF, IC1-DM1, and IC1-DM4, by hydrophobic interaction chromatography (HIC). [Figure 2C] Figure 2C: In vitro cytotoxicity of four ICAM1 ADCs against a panel of four human TNBC cell lines (MDA-MB-436, MDA-MB-468, MDA-MB-157, and MDA-MB-231) and two non-neoplastic cell lines (MCF10A and HEK293).

[0018] [Figure 3A-B] Figures 3A-3F show the tumor specificity and biodistribution of ICAM1 antibodies in nude mice. Figure 3A: Schematic design of TNBC biodistribution in an immunocompromised nude mouse model. Figure 3B: In vivo NIR fluorescence images of nude mice 48 hours after administration of IgG-Cy5.5, IC1-Cy5.5, or IC1-MMAE-Cy5.5 (n=5 per group). [Figure 3C-D] Figure 3C: Quantified MDA-MB-436 tumor accumulation of IgG-Cy5.5, IC1-Cy5.5, or IC1-MMAE-Cy5.5. *p<0.05, **p<0.01, NS: not significant. Figure 3D: Ex vivo NIR fluorescence images of MDA-MB-436 tumors treated with IgG-Cy5.5, IC1-Cy5.5, or IC1-MMAE-Cy5.5. [Figure 3E-F] Figure 3E: Representative ex vivo NIR fluorescence images of six major organs including brain (B), lung (LU), heart (H), liver (L), spleen (S), and kidney (K). Figure 3F: Quantified normal organ distribution of IgG-Cy5.5, IC1-Cy5.5, or IC1-MMAE-Cy5.5 (n=5).

[0019] [Figure 4A]Figures 4A-4D show the tumor specificity and biodistribution of ICAM1 antibodies in BALB / c mice. Figure 4A: Schematic design of tumor biodistribution in an immunocompetent BALB / c mouse model. [Figure 4B] FIG. 4B: Ex vivo NIR fluorescence images of 4T1 tumors and six normal organs treated with IgG-Cy5.5 and IC1-Cy5.5 (anti-mouse) (n=7 per group). [Figure 4C] Figure 4C: Quantified accumulation of IgG-Cy5.5 and IC1-Cy5.5 (anti-mouse) in 4T1 tumors and normal organs. **p<0.01, ***p<0.001. NS: not significant. [Figure 4D] FIG. 4D: Circulating leukocyte uptake of IgG-Cy5.5 and IC1-Cy5.5 (anti-mouse) quantified by flow cytometry. NS: not significant.

[0020] [Figure 5A-B] Figures 5A-5D show that ICAM1 ADC eradicates standard and late stage TNBC tumors in vivo. Figure 5A: Schematic design of the in vivo efficacy of ICAM1 ADC in standard and late stage orthotopic TNBC model. Figure 5B: Images of orthotopic MDA-MB-436 tumors excised from mice treated with PBS (sham), free Dox, ICAM1 antibody (IC1 Ab), IC1-MMAF, or IC1-MMAE at standard stage (n=7-10 per group). [Figure 5C-D]FIG 5C: Tumor progression in standard phase (left) was monitored by tumor volume measurement using calipers. Tumor mass (center) at standard phase endpoint (day 24) was quantified by weight. Mouse body weights in standard phase receiving PBS (sham), free Dox, IC1 Ab, IC1-MMAF, or IC1-MMAE (right). FIG 5D: Tumor progression in late phase receiving PBS (sham), IC1-MMAF, or IC1-MMAE (left) was monitored by tumor volume. Tumor mass (center) at late phase endpoint (day 34) was quantified by weight. Mouse body weights in late phase receiving PBS (sham), IC1-MMAF, or IC1-MMAE (right).

[0021] [Figure 6A] Figures 6A-6G show that ICAM1 ADC eradicates refractory TNBC tumors in vivo. Figure 6A: Schematic design of the in vivo efficacy of ICAM1 ADC in a refractory TNBC mouse model. [Figure 6B-D] Figure 6B: Images of orthotopic MDA-MB-231 tumors excised from mice treated with PBS (sham), IC1-MMAF, or IC1-MMAE. Figure 6C: Refractory tumor progression (left) receiving PBS (sham), IC1-MMAF, or IC1-MMAE was monitored by tumor volume measurement using calipers. Tumor mass (center) at endpoint (day 24) was quantified by weight. Mouse body weight quantified during administration of PBS (sham), free Dox, IC1 Ab, IC1-MMAF, or IC1-MMAE (right). Figure 6D: Schematic design of dose-dependent efficacy of IC1-MMAE in an orthotopic TNBC tumor model. [Figure 6E-F] Figure 6E: Images of orthotopic MDA-MB-436 tumors excised from mice treated with PBS (sham) or IC1-MMAE at three different dosages. Figure 6F: Tumor progression (left) was monitored by tumor volume receiving PBS (sham) or IC1-MMAE at different dosages. Tumor mass (center) at the endpoint (day 24) was quantified by weight. Quantified mouse body weight (right) during administration of IC1-MMAE at different dosages. [Figure 6G] FIG. 6G: Chronic hepatotoxicity and nephrotoxicity of IC1-MMAE were analyzed by blood chemistry.

[0022] [Figure 7A] Figures 7A and 7B show the MTD determination of IC1-MMAE. Figure 7A: Schematic design of the maximum tolerated dose of IC1-MMAE in an immunocompetent BALB / c mouse model. [Figure 7B] FIG. 7B: Mouse body weight quantified during the MTD study (n=10 per group).

[0023] [Figure 8] Figure 8 shows selective elimination of non-TNBC human cancer cells by ICAM1 ADCs. In vitro cytotoxicity of four ICAM1 ADCs (ICAM1-DM4, ICAM1-DM1, ICAM1-MMAE, and ICAM1-MMAF) was tested against a panel of four non-TNBC ICAM1-overexpressing human cancer cell lines: Du145 (prostate cancer), Caov3 (ovarian cancer), A375 (melanoma), and PC9 (lung cancer). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Antibody-drug conjugates (ADCs) I.ICAM1 antibody Antibody-drug conjugates (ADCs) are a class of immunotherapeutic agents that include an antibody conjugated to a drug. The ADCs of the present disclosure can target cells expressing ICAM1. ICAM1 is a cell surface glycoprotein that has been shown to bind to integrins of type CD11a / CD18 or CD11b / CD18 and has been implicated in mediating cell-cell interactions and promoting leukocyte endothelial transmigration. ICAM1 is also referred to as ICAM-1, BB2, cluster of differentiation 54 (CD54), and P3.58.

[0025] Non-limiting examples of amino acid sequences encoding ICAM1 include UniProtKB accession numbers P13597 and P05362. UniProtKB accession number P13597 encodes ICAM1 from Mus Musculus and has the following sequence: (Sequence number 1).

[0026] UniProtKB accession number P05362 encodes ICAM1 from Homo Sapiens and has the following sequence: (Sequence number 2).

[0027] In some embodiments, the ICAM1 protein comprises a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1 or SEQ ID NO:2. Additional ICAM1 proteins are also known and may be identified using publicly available databases, including, for example, GenBank. The ICAM1 protein may be from any species, including homo sapiens.

[0028] The antibody of the present disclosure is capable of binding to ICAM1. In some embodiments, the ICAM1 antibody is a monoclonal antibody. In some embodiments, the ICAM1 antibody is a polyclonal antibody. In some embodiments, the ICAM1 antibody is a mouse antibody. In some embodiments, the ICAM1 antibody is a humanized antibody. In some embodiments, the ICAM1 antibody is a chimeric antibody.

[0029] Non-limiting examples of ICAM1 antibodies include clone HCD54 ("HCD54" available from BioLegend, catalog # 322702), UV3, RR1.1, R6.5 (BIRR-1 or enlimomab available from Thermo Fisher Scientific, catalog # BMS1011), and BI-505. R6.5 (enlimomab) is, by way of example, a monoclonal mouse antibody produced by ATCC HB-9580 hybridoma cells, as described in U.S. Patent No. 5,324,510, which is incorporated herein by reference.

[0030] UV3 is a monoclonal antibody that has been shown to bind to ICAM-1 on myeloma cells. In some embodiments, the ICAM1 antibody is a F(ab)'2 fragment of UV3. See, for example, Huang et al., Hybridoma. 1993 Dec;12(6):661-75; and Coleman et al., J Immunother. 2006 Sep-Oct;29(5):489-98, each of which is incorporated herein by reference. RR1.1 is a monoclonal ICAM1 antibody. See, for example, Rothlein and Springer, 1986 J. Exp. Med. 163, 1132-1149, each of which is incorporated herein by reference. HCD54 is a monoclonal ICAM1 antibody. BI-505 is a fully human ICAM1 monoclonal antibody. See, e.g., Hansson et al., Clin Cancer Res. 2015 Jun 15;21(12):2730-6, which is incorporated herein by reference.

[0031] The term "bind" refers to the association of two entities (e.g., two proteins) that have an affinity (KD) between them of <10 -4 M, <10 -5 M, <10 -6 M, <10 -7 M, <10 -8 M, <10 -9 M, <10 -10 M, <10 -11 M, or <10 -12 M. Those skilled in the art are familiar with how to assess the affinity of two entities (e.g., two proteins).

[0032] The term "antibody" encompasses whole antibodies (immunoglobulins with two heavy and two light chains), antibody mimetics, and antibody fragments. "Immunoglobulins (Ig)" are large Y-shaped proteins produced primarily by plasma cells that are used by the immune system to neutralize foreign substances (e.g., pathogens such as bacteria and viruses). Antibodies may be classified as IgA, IgD, IgE, IgG, and IgM. "Antibody fragments" encompass any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In some embodiments, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each of the VH and VL is composed of 3 CDRs and 4 FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In some embodiments, the antibody is an immunoglobulin (Ig) monomer. The antibody may be a polyclonal or monoclonal antibody.

[0033] In some embodiments, the antibody is a heterotetrameric glycoprotein composed of two identical L chains and two H chains (IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic 4-chain units and J chains). For IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. At the N-terminus, each H chain has a variable domain (VH) followed by three constant regions (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CH1). Certain amino acid residues are believed to form an interface between the variable domains of the light and heavy chains. The pairing of a VH and a VL together forms a single antigen-binding site. For non-limiting examples of the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6 (incorporated herein by reference). In some embodiments, the antibody is an IgG.

[0034] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant regions. Depending on the amino acid sequence of the constant region (CH) of their heavy chains, immunoglobulins can be assigned to various classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; by way of example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0035] The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions", each 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs (mostly adopting a β-sheet configuration), connected by three hypervariable regions, which form loops that connect to, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs with the hypervariable regions from the other chain and contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

[0036] In some embodiments, the antibody is a monoclonal antibody. A "monoclonal antibody" is an antibody obtained from a substantially homogeneous antibody population, i.e., the population including the individual antibodies is identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic, animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, e.g., in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), which are incorporated herein by reference.

[0037] The monoclonal antibodies described herein encompass "chimeric" antibodies, as well as fragments of such antibodies (so long as they exhibit the desired biological activity), in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or within a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or within another antibody class or subclass (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human animal of the primate order (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0038] In some embodiments, the antibody is a polyclonal antibody. A "polyclonal antibody" is a mixture of different antibody molecules that react with more than one immunogenic antigenic determinant. Polyclonal antibodies may be isolated or purified from mammalian blood, secretions, or other body fluids, or from eggs. Polyclonal antibodies may also be recombinant. Recombinant polyclonal antibodies are polyclonal antibodies produced by the use of recombinant technology. Recombinantly produced polyclonal antibodies often contain a high concentration of different antibody molecules, all or a majority of which (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or more) exert the desired binding activity against an antigen composed of more than one epitope.

[0039] In some embodiments, antibodies are "humanized" for use in humans (e.g., as therapeutic agents). "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate animal that has the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody comprises substantially all of at least one variable domain, typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. Optionally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0040] In some embodiments, the antibody is an "antibody fragment" that contains the antigen-binding portion of a full-length ICAM1 antibody. In some embodiments, the antibody is a single-domain heavy chain antibody. In some embodiments, the antibody is a single-domain light chain antibody. The antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, which consists of the VH domain (e.g., as described in Ward et al., (1989) Nature 341:544-546, incorporated herein by reference); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to form a monovalent molecule as a single protein chain that pairs the VL and VH regions (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, which are incorporated herein by reference). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0041] In some embodiments, the antibody fragment may be an Fc fragment, an Fv fragment, or a single-change Fv fragment. The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by the sequences in the Fc region, which is also the portion recognized by the Fc receptor (FcR) found on certain types of cells.

[0042] Fv fragment is the minimum antibody fragment that contains the complete site of recognizing and binding antigen. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in tight non-covalent association. From these folding, the two domains generate six highly variable loops (three loops from each of H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, but with a lower affinity than the entire binding site.

[0043] Single-chain Fv, also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain that are connected to form a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the desired structure for antigen binding (e.g., as described in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995 (incorporated herein by reference)). In some embodiments, the antibody is a dimerized scFV (diabody), an scFV trimer (triabody), or an scFV tetramer (tetrabody).

[0044] The antibodies of the present disclosure include antibody mimetics, including affibody molecules. Affibodies are small proteins that contain a three-helix bundle that function as antigen-binding molecules (for example, antibody mimetics). In general, affibodies are approximately 58 amino acids in length and have a molar mass of approximately 6 kDa. Affibody molecules with unique binding properties are obtained by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. A specific affibody molecule that binds to a desired target protein can be isolated from a pool (library) containing billions of different variants using methods such as phage display.

[0045] In some embodiments, the ICAM1 antibody binds to an epitope present in the extracellular portion of ICAM1. The "extracellular portion" of ICAM1 refers to the portion of ICAM1 that is outside the cytosol and on the cell surface, as opposed to the portion that is inside the cytosol or embedded in the plasma membrane of the cell. The extracellular portion of ICAM1 typically comprises the glycosylated amino-terminal portion of the protein that mediates cell-cell interactions and promotes migration of leukocytes to the endothelium.

[0046] Methods for producing antibodies (e.g., monoclonal or polyclonal antibodies) are known in the art. For example, polyclonal antibodies may be prepared by immunizing an animal (preferably a mammal) with a selected allergen, followed by isolation of antibody-producing B lymphocytes from the blood, bone marrow, lymph nodes, or spleen. Alternatively, antibody-producing cells may be isolated from the animal and exposed in vitro to the allergen (to which the antibodies will be raised). The antibody-producing cells may then be cultured to obtain an antibody-producing cell population, optionally after fusion to an immortalized cell line such as a myeloma. In some embodiments, B lymphocytes as starting material may be isolated from tissues of allergic patients to generate fully human polyclonal antibodies. Antibodies may be produced in mice, rats, pigs (swine), sheep, bovine material, or other transgenic animals with human immunoglobulin genes as starting material to generate fully human polyclonal antibodies. In some embodiments, mice or other animals transgenic for human immunoglobulin genes (e.g., as disclosed in U.S. Pat. No. 5,939,598) may be immunized to stimulate the in vivo production of specific antibodies and antibody-producing cells prior to preparation of polyclonal antibodies from the animals by extraction of B lymphocytes or purification of polyclonal serum.

[0047] Monoclonal antibodies are typically produced by cell culture involving fusing myeloma cells with mouse spleen cells immunized with the desired antigen (i.e., hybridoma technology). The mixture of cells is diluted and clones are grown from a single parent cell on a microtiter well. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (in tests such as ELISA or antigen microarray assays) or immunodot blotted. The most productive and stable clones are then selected for further use.

[0048] II. Drugs Suitable drugs for use in ADCs include agents that are therapeutically active against triple-negative breast cancer (TNBC). Non-limiting examples of drugs include chemotherapeutic agents. In some cases, the drug is a small molecule. In some embodiments, the drug is a cytotoxic small molecule. In some embodiments, the drug is a cytostatic small molecule.

[0049] Non-limiting examples of drugs suitable for use in ADCs include N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)mertansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin, paclitaxel, everolimus, fluorouracil (5-FU), gemcitabine, gemcitabine hydrochloride, mitomycin C, and derivatives thereof. In some embodiments, the drug is maytansine or an analog thereof. In some embodiments, the drug is DM1. DM1 is a cytotoxic maytansine analog that has been shown to inhibit tubulin polymerization. In some embodiments, the maytansine analog is DM4.

[0050] The term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Typically, small molecules are organic compounds (e.g., containing carbon). Small molecules may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, heterocyclic rings, and the like). In some embodiments, the molecular weight of a small molecule is about 1,000 g / mol or less, about 900 g / mol or less, about 800 g / mol or less, about 700 g / mol or less, about 600 g / mol or less, about 500 g / mol or less, about 400 g / mol or less, about 300 g / mol or less, about 200 g / mol or less, or about 100 g / mol or less. In some embodiments, the molecular weight of a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol, and combinations of the above ranges are also possible (e.g., at least about 200 g / mol and up to about 500 g / mol).

[0051] Any known chemotherapeutic agent may be used as the drug in the ADCs described herein. Non-limiting exemplary chemotherapeutic agents include the following: actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0052] III. Linker One or more drugs may be conjugated to the ICAM1 antibody using techniques known in the art. In some embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) drugs are conjugated to the ICAM1 antibody. The ratio of ICAM1 antibody to drug in the ADC may be 1:1 to 1:10 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10). In some embodiments, the ratio of ICAM1 antibody to drug in the ADC is 1:4.

[0053] The ICAM1 antibody may be conjugated to a second entity, either directly or via a linker. As used herein, "conjugated" or "attached" means that the two entities are linked, preferably through a covalent bond or with sufficient affinity, such that the therapeutic or diagnostic benefit of the association between the two entities is realized. In some embodiments, the linker conjugates the ICAM1 antibody to a drug in the ADC. The N-terminus or C-terminus of the ICAM1 antibody may be conjugated to the drug. In some embodiments, the linker may be used to conjugate the ICAM1 antibody to an imaging agent. The N-terminus or C-terminus of the ICAM1 antibody may be conjugated to the imaging agent.

[0054] In some embodiments, the linker is a cleavable linker. As used herein, a cleavable linker is capable of releasing a conjugated moiety in response to a stimulus. In some embodiments, the stimulus is a physiological stimulus. Non-limiting examples of stimuli include the presence of an enzyme, acidic conditions, basic conditions, or reducing conditions. For example, cleavable linkers include peptide linkers, β-glucuronide linkers, glutathione-sensitive linkers (or disulfide linkers), and pH-sensitive linkers. In some embodiments, the pH-sensitive linker is cleaved at a pH between 5.0 and 6.5, or between 4.5 and 5.0. In some embodiments, the pH-sensitive linker is not cleaved when the pH is between 7 and 7.5. In some embodiments, the pH-sensitive linker is not cleaved when the pH is between 7.3 and 7.5. In some embodiments, the cleavable linker is a protease-sensitive linker.

[0055] Examples of cleavable linkers include N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), Sulfo-SPDB, valine-citrulline dipeptide (Val-cit), acetylbutyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal. In some embodiments, the cleavable linker is a maleimidocaproyl (MC) or Mal-EBE-Mal linker. See, e.g., Donaghy, MAbs. 2016 May-Jun;8(4):659-71, incorporated herein by reference.

[0056] In some embodiments, the linker is non-cleavable. In some embodiments, the non-cleavable linker is a linker that is not cleaved in the systemic circulation of a subject. In some embodiments, the non-cleavable linker is a linker that is resistant to protease cleavage. Non-cleavable linkers include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidomethylcyclohexane-1-carboxylate (MCC), and MC-VC-PAB. In some embodiments, the non-cleavable linker is MC-VC-PAB.

[0057] Any of the antibody-drug conjugates may be synthesized using methods known in the art, see, for example, Yao et al., Int J Mol Sci. 2016 Feb 2;17(2).pii:E194.

[0058] ADCs comprising an ICAM1 antibody conjugated to a drug are also advantageous for use in therapy, in part because the drug (e.g., a chemotherapeutic drug) is toxic and causes severe side effects. By conjugating a drug (e.g., DM1) to an ICAM1 antibody, the toxicity of the ADC may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the drug in its free form.

[0059] Other ICAM1 antibody conjugates Any of the ICAM1 antibodies and / or ADCs of the present disclosure may be conjugated to an imaging agent that may be useful for predicting the therapeutic sensitivity of TNBC subjects. For example, imaging agents for computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and endoscopic detection (e.g., endoscopic ultrasound) may be used, which may include contrast agents. For example, see Bird-Lieberman et al., Nat Med. 2012; 18(2): 315-21; Van den Brande et al., Gut. 2007; 56(4): 509-17, each of which is incorporated herein by reference. In some embodiments, the imaging agent is administered as a salt. In some embodiments, the imaging agent is a gadolinium-based MRI contrast agent. For example, the imaging agent may be gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA or DTPA-Gd). See, e.g., Carr et al., AJR Am J Roentgenol. 1984 Aug;143(2):215-24.

[0060] One or more imaging agents may be conjugated to the ICAM1 antibody or ADC described herein using techniques known in the art. In some embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) imaging agents are conjugated to the ICAM1 antibody. The ratio of ICAM1 antibody or ADC to imaging agent may be 1:1 to 1:10 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10). In some embodiments, the ratio of ICAM1 antibody or ADC to imaging agent is 1:4. Any of the linkers disclosed herein may be used to conjugate an imaging agent to the ICAM1 antibody or ADC described herein. The imaging agent may be visualized by a suitable detection method (eg, by CT, PET, MRI, ultrasound, and / or endoscopic detection).

[0061] Pharmaceutical compositions and their uses Compositions comprising any of the ADCs or other ICAM1 antibody conjugates disclosed herein are encompassed by the present disclosure. In some embodiments, the compositions are formulated as pharmaceutical compositions for administration to a subject.

[0062] The subject may have, be suspected of having, or be at risk of having triple-negative breast cancer (TNBC). Breast cancer is classified based on the receptor proteins that are or are not expressed on the cell surface of breast cancer cells. TNBC cells are characterized as breast cancer cells that lack cell surface expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). In contrast, ER+ breast cancer cells express estrogen receptor on their cell surface, while HER2+ breast cancer cells express HER2 on their cell surface.

[0063] TNBC may also be stratified based on whether the cancer has metastasized. TNBC may be classified as stage 0 (cancer in situ), stage I, stage II, stage III, stage IV, or stages therewithin (e.g., stage IIA, stage IIB, etc.). A non-limiting staging method is the TNM system, which evaluates the extent of the tumor (T), the spread of the cancer to nearby lymph nodes (N), and whether the cancer has spread to distant sites (M). The various levels of T, N, and M (e.g., Table 1) may then be used to determine the stage of TNBC (e.g., Table 2). Tables 1-2 show the TNBC classification based on the 8th edition of the AJCC / UICC TNM staging system and as described by Cong et al. Sci Rep. 2018 Jul 10;8(1):10383.

[0064] Table 1. Non-limiting examples of TNM staging definitions [Table 1]

[0065] Table 2. TNBC Staging Levels [Table 2]

[0066] In some embodiments, the subject may be suspected of having or at risk of having cancer other than TNBC. Non-limiting examples of other cancers include breast cancer, prostate cancer, ovarian cancer, melanoma, lung cancer, and pancreatic cancer. In some embodiments, breast cancer is not TNBC. Other cancers may also be stratified based on whether the cancer has metastasized or not, and may be classified into stage 0 (cancer in situ), stage I, stage II, stage III, stage IV, or intermediate stages (e.g., stage IIA, stage IIB, etc.). Other cancers may also be classified using the TNM staging method, in which various levels of T, N, and M are used to determine the stage of cancer (e.g., Tables 1 and 2).

[0067] In some embodiments, any of the pharmaceutical compositions disclosed herein comprising an imaging agent is administered in an effective amount to a subject to determine the level of ICAM1 in a tumor of a TNBC subject or another cancer subject (e.g., CT, PET, MRI, and endoscopic detection (e.g., endoscopic ultrasound)). The imaging methods for determining the level of ICAM1 described herein are advantageous compared to traditional methods (e.g., biopsy and analysis of tissue obtained from the biopsy). The imaging methods (e.g., MRI) are non-invasive and provide a comprehensive view of the tumor for ICAM1 levels, thereby providing a more accurate assessment for predicting outcome and / or responsiveness to tumor treatment (e.g., treatment with an ICAM1 antibody or an ADC comprising an ICAM1 antibody).

[0068] In some embodiments, the level of ICAM1 is detected in a TNBC subject or another cancer subject administered a pharmaceutical composition of the present disclosure comprising an ICAM1 antibody and an imaging agent. In some embodiments, the level of ICAM1 detected in the subject's tumor is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% higher than the control. In some embodiments, the level of ICAM1 detected in the subject's tumor is substantially similar to the control.

[0069] In some embodiments, the control is a subject with a tumor having a known level of ICAM1. In some embodiments, the control is the level of ICAM1 in breast tissue of a subject without a tumor. In some embodiments, the control is a subject with a tumor having a low level of ICAM1. In some embodiments, the low level of ICAM1 is undetectable. In some embodiments, the control is a subject with a tumor having a high level of ICAM1. In some embodiments, the high level of ICAM1 is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% higher than the level of ICAM1 detected in breast tissue of a healthy subject.

[0070] In some embodiments, the level of ICAM1 detected in a tumor using the methods disclosed herein predicts a TNBC subject or another cancer subject that will respond to treatment with an ICAM1 antibody or an antibody drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to a drug. In some embodiments, a higher level of ICAM1 detected in a tumor predicts that the tumor will be more responsive to treatment with an ICAM1 antibody or an ADC disclosed herein, as compared to a tumor in a subject with a lower level of ICAM1. In some embodiments, subjects with higher levels of ICAM1 in their tumors are at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% more responsive to treatment with a composition comprising an ICAM1 antibody (e.g., an ICAM1 ADC, and / or an ICAM1 antibody not conjugated to a drug) compared to subjects with lower levels of ICAM1 in their tumors. In some embodiments, the methods disclosed herein comprise administering an ICAM1 antibody or an ADC antibody disclosed herein after identifying the subject as responsive.

[0071] In some embodiments, the level of ICAM1 detected in a tumor using the methods disclosed herein is indicative of the stage of the cancer, hi some embodiments, the level of ICAM1 detected in a tumor is indicative of stage 0, stage I, stage II, stage III, or stage IV.

[0072] Without being bound to a particular theory, in some embodiments, administration of an ICAM1 antibody conjugated to an imaging agent or an ICAM1 ADC conjugated to an imaging agent may serve the dual purpose of visualizing the tumor and treating the tumor.

[0073] In some embodiments, administration of an ICAM1 antibody and / or an ADC comprising an ICAM1 antibody, or a pharmaceutical composition thereof, inhibits tumor growth. In some embodiments, administration of an ICAM1 antibody and / or an ADC comprising an ICAM1 antibody, or a pharmaceutical composition thereof, results in tumor regression. In some embodiments, administration of an ICAM1 antibody and / or an ADC comprising an ICAM1 antibody, or a pharmaceutical composition thereof, reduces the size of a tumor by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% compared to a control. In some embodiments, the control is a subject not treated with a composition comprising an ICAM1 antibody.

[0074] In some embodiments, administration of an ICAM1 antibody and / or an ADC disclosed herein comprising an ICAM1 antibody, or a pharmaceutical composition thereof, reduces proliferation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% higher than a control. In some embodiments, proliferation is measured using Ki67 staining. In some embodiments, the control is a subject not treated with a composition comprising an ICAM1 antibody.

[0075] In some embodiments, administration of an ICAM1 antibody and / or an ADC disclosed herein comprising an ICAM1 antibody, or a pharmaceutical composition thereof, reduces tumor metastasis by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% compared to a control. In some embodiments, the control is a subject not treated with a composition comprising an ICAM1 antibody.

[0076] In some embodiments, administration of an ICAM1 antibody and / or an ADC disclosed herein comprising an ICAM1 antibody, or a pharmaceutical composition thereof, does not reduce the viability of healthy cells. In some embodiments, administration of an ADC disclosed herein or a pharmaceutical composition comprising an ADC may cause the effective amount (e.g., concentration) of the drug to be lower than if the drug was not conjugated to the ICAM1 antibody. In some embodiments, the effective amount of the drug is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000%, compared to administration of the drug alone.

[0077] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable carrier" may be a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the agent of interest from one organ (or part of the body) to another organ (or part of the body). Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient's tissues (e.g., physiological compatibility, sterility, physiological pH, etc.). The term "carrier" refers to a natural or synthetic, organic or inorganic ingredient that is combined with the active ingredient to facilitate its application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.Some examples of materials which may serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) glycerin, soybean oil, and the like. Examples of suitable non-toxic compatible materials include polyols, such as sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible materials employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.

[0078] Pharmaceutical compositions may conveniently be in unit dosage form and may be prepared by any method well known in the pharmaceutical field.The term "unit dose" when used in relation to pharmaceutical compositions of the present disclosure refers to a physically discrete unit suitable for subject as a unitary dosage, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in association with required diluent; i.e., carrier or vehicle.

[0079] The formulation of the pharmaceutical composition may depend on the route of administration. Suitable injectable preparations for parenteral administration or intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, which may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3 propanediol or 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution (USP), and isotonic sodium chloride solution. In addition, sterile fixed oils are also conventionally employed as solvents or suspending media. For this purpose, any sterile fixed oil may be employed, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are also used in the preparation of injectables. The injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other injectable sterile medium prior to use.

[0080] Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or emulsion.

[0081] In some embodiments, pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (for example, a 0.2 micron membrane). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known, including, for example, phenol and ascorbic acid. Pharmaceutical compositions will usually be stored in lyophilized form or as aqueous solution, where they are highly stable against thermal and oxidative denaturation. The pH of the preparation will typically be about 6 to 8, although higher or lower pH values ​​may also be appropriate in some cases.

[0082] "Therapeutically effective amount" or "effective amount" as used herein refers to the amount of each therapeutic agent of the present disclosure (e.g., a therapeutic agent for treating any of the cancers described herein) required to produce a therapeutic effect on a subject (either alone or in combination with one or more other therapeutic agents). As recognized by those skilled in the art, the effective amount will vary depending on the specific disease being treated, the severity of the disease, the parameters of the individual subject (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concomitant treatment (if any), the specific route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dose of the individual components or combinations thereof be used, i.e., the highest safe dose according to sound medical judgment. However, it will be understood by those skilled in the art that a subject may require a lower or tolerable dose for medical reasons, psychological reasons, or any other reason of fact.

[0083] Empirical considerations such as half-life will generally contribute to the determination of dosage. For example, therapeutic agents compatible with the human immune system, such as polypeptides containing regions from humanized antibodies or fully human antibodies, may be used to extend the half-life of the polypeptide and prevent it from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment, and is generally based on (but not necessarily based on) treating and / or suppressing and / or recovering and / or delaying disease. Alternatively, sustained continuous release formulations of polypeptides may also be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0084] In some embodiments, dosing is every day, every other day, every 3 days, every 4 days, every 5 days, or every 6 days.In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer.The progress of this treatment is easily monitored by conventional techniques and assays.Dosing regimen (including anticancer drug used) can vary over time.

[0085] In some embodiments, for a normal weight adult subject, a dose ranging from about 0.01 to 1000 mg / kg may be administered. In some embodiments, the dose is between 1-200 mg. The specific dosing regimen, i.e., dose, timing, and repetition, will depend on the specific subject and the subject's medical history, as well as the characteristics of the anticancer agent, such as the half-life of the anticancer agent, and other considerations well known in the art.

[0086] For purposes of this disclosure, the appropriate dosage of a therapeutic agent as described herein will depend on the particular agent (or composition thereof) employed, the formulation and route of administration, the type and severity of the disease, whether the anti-cancer agent is administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the anti-cancer agent until a dosage is reached that achieves the desired result. Administration of one or more anti-cancer agents can be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the anti-cancer agent may be essentially continuous over a preselected period of time, or may be a series of doses spaced apart, for example, before, during, or after the onset of the disease.

[0087] As used herein, the term "treating" refers to the application or administration of an anti-cancer agent to a subject in need thereof. A "subject in need thereof" refers to an individual having a disease, a symptom of a disease, or a predisposition to a disease for the purpose of curing, heal, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, the symptom of a disease, or the predisposition to a disease.

[0088] A "subject" to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., a minor, a child, or an adolescent), or an adult subject (e.g., a young adult, a middle-aged adult, or an older adult)), or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a rodent (e.g., a mouse or a rat), a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cattle, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, such as a chicken, a duck, a goose, or a turkey)). The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic or genetically modified animal.

[0089] The methods described herein may be used to treat ICAM1-expressing cancer.In some embodiments, the ICAM1-expressing cancer is breast cancer, prostate cancer, ovarian cancer, melanoma or lung cancer.In some embodiments, the ICAM1-expressing cancer is not TNBC.

[0090] In some embodiments, the subject is a companion animal (pet or service animal). "Companion animal" as used herein refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include the following: rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human animals of the primate order.

[0091] Alleviating a disease (e.g., cancer) includes delaying the onset or progression of the disease or reducing disease severity. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone the progression of the disease. The delay can be for a variable amount of time depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or a method of delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of the symptoms within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0092] "Onset" or "progression" of a disease refers to early signs and / or subsequent progression of the disease. Onset of a disease can be detectable and assessed using standard clinical techniques as known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a disease includes initial onset and / or recurrence.

[0093] Conventional methods known to those skilled in the art of medicine can be used to administer the pharmaceutical composition to a subject depending on the type of disease or site of disease to be treated. The pharmaceutical composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, substernal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the pharmaceutical composition is administered via intravenous injection or infusion. In addition, it can be administered to a subject via an injectable depot route of administration, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods. In some embodiments, the pharmaceutical composition is administered via injection. In some embodiments, the injection is an intravenous injection or intratumoral injection.

[0094] example Example 1: Development of antibody-drug conjugates (ADCs) targeting ICAM1 introduction Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). Late-stage and refractory TNBC poses a major challenge to improving clinical outcomes for breast cancer patients. It is estimated that more than 28,000 patients will be diagnosed with TNBC in the United States in 2021, representing 10-15% of breast cancer incidence. 1~3 TNBC is more common in non-Hispanic black women, women younger than 40 years, and women with early-onset breast cancer 1 or 2 (BRCA1 / 2) gene mutations. 4,5 Moreover, patients with late-stage or refractory TNBC tumors respond poorly to first-line chemotherapy and do not respond to established hormone- and HER2-targeting therapies due to lack of expression of these molecular targets, thereby dramatically worsening their poor clinical outcomes. 2,3,6The prognosis for TNBC patients remains the poorest of all breast cancer patients, with current 5-year survival rates of less than 77%, significantly lower than the >90% survival rates for patients with other types of breast cancer. 7~9 .

[0095] Although several second-line therapies, including poly(ADP)-ribose polymerase (PARP) inhibitors (e.g., olaparib and talazoparib) and immune checkpoint inhibitors (e.g., atezolizumab and pembrolizumab), have been approved for the treatment of TNBC, the clinical benefit of these therapies is limited to a small subset of TNBC patients (up to 20-30%) due to limited BRCA1 / 2 mutation rates or PD-L1 expression. 10~12 Novel therapies targeting TNBC with distinct mechanisms of action that may act synergistically with these approved modalities are likely to further increase clinical efficacy. 13~15 Antibody-drug conjugates (ADCs) are an emerging class of immunochemotherapeutics that feature the structure of a monoclonal antibody conjugated to a cytotoxic agent via a chemical linker. The monoclonal antibody of an ADC serves as a tumor-homing ligand to guide the conjugated drug, thereby selectively eradicating antigen-overexpressing tumors while sparing normal tissues. Compared with T-cell immunotherapy (e.g., chimeric antigen receptor-T cells (CAR-T) or immune checkpoint blockade) or nanomedicine (e.g., liposomes), ADCs feature superior tumor tissue penetration due to their ultrasmall size (<10 nm), ~1,000 times smaller than that of T cells, creating an attractive opportunity to increase drug delivery into tumors. ADCs have demonstrated promising clinical efficacy in various cancers, including invasive solid tumors that respond poorly to T-cell immunotherapy. 13 As an illustration, sacituzumab govitecan, an ADC targeting trophoblast antigen 2 (TROP2), has been approved to treat patients with refractory TNBC with a response rate of ~33% and a median progression-free survival of 5.5 months. 16Given the poor prognosis and disappointing efficacy of current TNBC treatment modalities, there remains an urgent and unmet need to identify new druggable targets and develop more effective ADCs for the treatment of TNBC.

[0096] ICAM1, also called CD54, is a transmembrane cell surface glycoprotein that regulates cell-cell adhesion during inflammatory injury, viral infection, and tumor development. 17 ICAM1 is aberrantly overexpressed in multiple types of cancer and is often associated with an invasive phenotype and poor prognosis. ICAM1 was previously identified as a TNBC cell surface marker through a randomized quantitative screen of G protein-coupled receptor (GPCR) signaling proteins. 18,19 ICAM1 was found to be highly abundant on the cancer cell surface of human TNBC tumors compared with normal breast tissue. 18~22 The NFkB pathway has also been implicated in aberrantly upregulated ICAM1 expression in cancer cells by binding to the ICAM1 promoter and causing its enhanced transcription. 23,24 ICAM1-neutralizing antibodies have been investigated as potential cancer therapeutics, however, blocking the ICAM1 signaling cascade alone has not produced satisfactory clinical efficacy. 25~27 Thus, as described herein, ICAM1 ADCs have been developed that induce potent and durable tumor regression in vivo. We hypothesized that rationally designed ICAM1 ADCs would precisely target and eradicate TNBC tumors while sparing healthy organs and tissues. To test this hypothesis, a panel of four ICAM1 ADCs with different chemical linkers and cytotoxic agents were constructed and evaluated in vitro against multiple TNBC cell types. The in vivo efficacy of these ADCs was also tested in a series of standard-stage, late-stage, and refractory TNBC models.

[0097] Results and Discussion ICAM1 was previously identified as a novel molecular target for TNBC by measuring protein expression using immunohistochemistry (IHC) in a cohort of 149 human breast tumor tissues along with 144 human normal tissues encompassing the breast and 19 other organs. 18 ICAM1 staining was found to be present at significantly higher levels in cancer cells of TNBC tumors compared to other breast cancer subtypes, and was completely absent in normal breast tissue. Genomic analysis was performed using the R2: Genomics Analysis and Visualization Platform ( hgserver1.amc.nl We further investigated the relationship between ICAM1 expression and more detailed characteristics of TNBC, including its molecular subtype, tumor differentiation grade, and oncogene mutation status, by querying ICAM1 mRNA expression in three breast cancer subtypes: ER-positive, HER2-positive, and TNBC (Figure 1A). Consistent with the pathological staining results, ICAM1 mRNA expression was quantitatively compared in three breast cancer subtypes: ER-positive, HER2-positive, and TNBC (Figure 1B). 18 In TNBC tumor tissues (n = 116), ICAM1 mRNA levels were significantly higher than those in HER2-positive (n = 40) and ER-positive (n = 808) breast tumors. Recently, TNBC has been characterized into four unique molecular subtypes: basal-like immunosuppressed (BLIS), basal-like immunoreactive (BLIA), luminal androgen receptor (L-AR), and mesenchymal (MES). 28Given that TNBC has been identified as a heterogeneous disease classified into three subtypes, ICAM1 mRNA levels were also assessed in these molecular subtypes. BLIA (n=54) showed the highest level of ICAM1 expression, followed by MES (n=47), L-AR (n=60), and BLIS (n=37). The correlation between ICAM1 expression and TNBC tumor differentiation grade was also analyzed, and ICAM1 expression was found to be significantly higher in poorly differentiated TNBC tumors (grade 3, n=108) compared to moderately differentiated tumors (grade 2, n=53). It is generally known that poorly differentiated TNBC tumors are strongly associated with a poorer prognosis, and thus ICAM1 may serve as a potential biomarker for predicting clinical outcomes in TNBC patients. The relationship between ICAM1 expression and cancer mutation status was also examined, and ICAM1 levels were significantly higher in patients with BRCA1 / 2 mutations or TP53 mutations, which occur in 20% and 80% of TNBC tumors, respectively. 29~31 ) was found to be positively correlated with

[0098] Given that TROP2 is a new clinically approved ADC target for the treatment of TNBC 16, which was selected as a positive control for the evaluation of potential ICAM1 ADCs. We examined the cell surface expression of ICAM1 and TROP2 in three established human TNBC cell lines (MDA-MB-436, MDA-MB-157, and MDA-MB-231) along with normal human breast epithelial MCF10A cells (Figure 1B). The cell surface expression of ICAM1 is 11- to 35-fold higher than TROP2 on two TNBC cell lines (MDA-MB-436 and MDA-MB-468) and comparable in the third cell line (MDA-MB-231). More importantly, ICAM1 surface expression on normal MCF10A cells is 7-fold lower than that of TROP2. These findings were confirmed by immunofluorescence (IF) staining of ICAM1 and TROP2 in the same cells (Figure 1C). The intracellular localization of ICAM1 is mainly on the plasma membrane of TNBC cells and is undetectable on the plasma membrane of normal MCF10A cells. These results provide evidence that ICAM1 is more highly overexpressed and more tumor-specific than TROP2 in human TNBC cells, which may potentially contribute to higher tumor specificity and fewer on-target / off-tumor adverse effects during in vivo delivery of ICAM1-targeting ADCs.

[0099] Cellular internalization is another important factor affecting the efficacy of ADCs. To determine whether ICAM1 ADCs can selectively enter TNBC cells and rapidly translocate into endosomes for the release of cytotoxic agents, the cellular internalization rate of ICAM1 antibody was examined in multiple TNBC cell lines as well as normal MCF10A cells. Confocal fluorescence images revealed that ICAM1 antibody was rapidly internalized by all three TNBC cell lines examined (MDA-MB-436, MDA-MB-157, and MDA-MB-231) after 1 hour of incubation (Figure 1D). The internalized ICAM1 antibody aggregated rather than dispersed in the cytoplasm of TNBC cells, indicating that they were translocated into endosomes / lysosomes by receptor-mediated endocytosis. 32,33 In contrast, no internalization was observed in normal MCF10A cells due to their lack of ICAM1 antigen expression. The cellular internalization rates of ICAM1 antibodies in the three TNBC cell lines were approximately 42-109-fold higher than those in MCF10A cells and approximately 21-129-fold higher than those observed with non-targeting IgG (Figure 1E). These results confirm that ICAM1 is highly overexpressed in TNBC cells, facilitating efficient cellular internalization of bound antibodies, and suggest that ICAM1 has outstanding potential utility as an ADC target for treating TNBC.

[0100] To identify the optimal ADC formulation for TNBC, a panel of ICAM1 ADCs was synthesized using a monoclonal human / mouse chimeric ICAM1 antibody (clone #R6.5). 34We designed and constructed ICAM1 ADCs by conjugating them with cytotoxic drugs (Figure 2A) to generate four different ADC combinations, namely, MC-Vc-Pab-MMAE, MC-MMAF, Mal-EBE-Mal-DM1, and Mal-EBE-Mal-DM4. The ICAM1 ADCs were named according to their antibody and conjugated drug as follows: ICAM1 antibody conjugated to MC-Vc-Pab-MMAE (IC1-MMAE); ICAM1 antibody conjugated to MC-MMAF (IC1-MMAF); ICAM1 antibody conjugated to Mal-EBE-Mal-DM1 (IC1-DM1); and ICAM1 antibody conjugated to Mal-EBE-Mal-DM4 (IC1-DM4). The ICAM1 antibody selection was based on the excellent human safety profile of the human / mouse chimeric ICAM1 antibody (clone #R6.5) previously determined in phase I / II clinical trials. 27,34 The ADC linker-drug selection was then examined. MC-VC-Pab-MMAE features an enzymatically cleavable linker that can be selectively cleaved by cathepsin B proteases in cellular endosomes / lysosomes, resulting in rapid drug release from the ADC antibody upon cellular internalization while maintaining the integrity of the ADC throughout blood circulation. 13~15 Other MC linkers and the Mal-EBE-Mal linker are non-cleavable, and therefore drug release is dependent on lysosomal degradation of the conjugated antibody. Both enzyme-cleavable and non-cleavable linkers are currently used in clinically approved ADCs to treat various cancers. 13~15 All four drugs utilized in the ICAM1 ADCs tested are potent microtubule inhibitors with cytotoxicity approximately 100-1,000-fold higher than standard-of-care chemotherapeutic drugs (e.g., paclitaxel or gemcitabine). 13~15MMAE, MMAF, and DM1 are clinically utilized ADC drugs, and DM4 is also being clinically investigated as an ADC drug to treat TNBC. However, it remains unclear which ADC linker-drug combination is most effective for TNBC treatment in the absence of randomized quantitative screens. Moreover, drug:antibody ratio (DAR), defined as the number of drug molecules conjugated per antibody, is another important factor that affects the efficacy and safety of ADCs in humans. A DAR value of 4.0 is well established in multiple clinically approved ADCs (e.g., trastuzumab emtansine and brentuximab vedotin), which are microtubule inhibitors that demonstrate excellent clinical efficacy and human safety profiles. 13~15 Therefore, the DAR of the constructed four ICAM1 ADCs was adjusted to a value equivalent to 4 by controlling the antibody / drug input ratio during ADC conjugation. The resulting DAR values ​​of the four ICAM1 ADCs were determined by hydrophobic interaction chromatography (HIC) as 3.96 for IC1-DM1, 3.83 for IC1-DM4, 3.92 for IC1-MMAE, and 4.09 for IC1-MMAF, respectively (Figure 2B).

[0101] The in vitro cytotoxicity of the four ICAM1 ADCs was then assessed against a panel of TNBC cell lines, and the half maximum inhibitory concentrations (IC50) were quantified using the Dojindo assay. Among the four ICAM1 ADCs, exposure to IC1-MMAE and IC1-MMAF elicited significantly higher in vitro potency compared to IC1-DM1 and IC-DM4 in three of the four TNBC cell lines tested (Figure 2C). The IC50s of IC1-MMAE and IC1-MMAF were determined to be 13.1 pM and 7.3 pM for MDA-MB-436, 250.0 pM and 68.7 pM for MDA-MB-468, and 221.7 pM and 116.0 pM for MDA-MB-157, respectively. It is noteworthy that MDA-MB-436 and MDA-MB-157 cells represent Caucasian and African-American TNBC cell lines, respectively. In contrast, the IC50 of IC1-DM1 and IC1-DM4 are at least 5-fold higher. Notably, the IC50 of IC-MMAE and IC-MMAF are approximately 1,000-fold lower than that of doxorubicin, the standard-of-care chemotherapy drug used for TNBC treatment. It was observed that MDA-MB-231 cells exhibited strong drug resistance to all four ICAM1 ADCs tested. MDA-MB-231 cells were also reported to be resistant to the TROP2 ADC sacituzumab govitecan, which is clinically used for TNBC. 35Due to this fact, this TNBC cell line was selected for the generation of an ADC-resistant TNBC model in the following animal studies. The cytotoxicity of ICAM1 ADCs was also evaluated in non-neoplastic MCF10A and HEK293 cells, both of which lack ICAM1 antigen expression. As expected, no cytotoxicity was observed in ICAM1-negative MCF10A and HEK293 cells until extremely high ADC concentrations of >6.7 nM were reached. This indicates that the efficacy of ADCs to eliminate TNBC cells is dependent on the cell surface expression of ICAM1. Together, these in vitro efficacy screening data support the notion that IC1-MMAE and IC1-MMAF are more potent ADC formulations than ICAM1-DM1 and ICAM1-DM4 against TNBC, and justify further study of these ADCs using in vivo TNBC models.

[0102] Next, to study the effect of different immune system backgrounds, the tumor specificity and biodistribution of ICAM1 ADCs were evaluated using both an immunocompromised nude mouse model bearing human TNBC tumors (MDA-MB-436) and an immunocompetent BALB / c mouse model bearing murine TNBC tumors (4T1). First, the nude mouse model was used to determine the tumor specificity of ICAM1 antibodies and ADCs in orthotopic human TNBC tumors (Figure 3A). Near-infrared fluorescent dye Cy5.5-labeled anti-human ICAM1 antibodies (IC1-Cy5.5) and IC1-MMAE (IC1-MMAE-Cy5.5) were administered intravenously and compared to nontargeted IgG conjugated with Cy5.5 (IgG-Cy5.5) at an equivalent dosage of 5 mg / kg. Nude mice were imaged by in vivo NIR fluorescence imaging 24 hours after injection. Mice treated with IC1-Cy5.5 and IC1-MMAE-Cy5.5 showed comparable intratumoral accumulation, approximately 5-fold higher than IgG-Cy5.5 (Figures 3B and 3D). These in vivo tumor accumulation data precisely matched the ex vivo NIR images of resected tumors (Figure 3C). These results suggest that IC1-Cy5.5 and IC1-MMAE-Cy5.5 selectively recognize and bind human TNBC tumors in vivo compared to non-targeted IgG-Cy5.5. The biodistribution of IC1-Cy5.5, IC1-MMAE-Cy5.5, and IgG-Cy5.5 was further examined in six major organs, namely brain, lung, heart, liver, spleen, and kidney. The liver and kidney were the main extratumoral accumulation sites of IC1-Cy5 and IC1-MMAE in tumor-bearing mice (Figures 3E and 3F). However, these are common extratumoral deposition sites and it was determined that any toxicity was well tolerated based on serum biomarkers in subsequent animal studies.

[0103] A potential concern with the use of ICAM1 as a drug target to develop ADCs targeted to TNBC is its potential on-target / off-tumor toxicity in normal organs and tissues that normally express ICAM1. In nude mouse models, anti-human ICAM1 antibodies cannot recognize the mouse ICAM1 antigen expressed in normal mouse organs due to lack of species cross-reactivity. To address this issue, biodistribution in normal organs was assessed using anti-mouse ICAM1 antibodies in an immunocompetent BALB / c mouse model bearing mouse 4T1 tumors (Figure 4A). Notably, unlike the immunocompromised nude mouse model, this biodistribution study was performed in immunocompetent BALB / c mice that feature an intact immune system that may more faithfully recapitulate the interaction between the TNBC tumor microenvironment and ICAM1 antibodies. Anti-mouse IC1-Cy5.5 maintains strong 4T1 tumor specificity in BALB / c mice and correlates well with anti-human IC1-Cy5.5 in nude mice (Figure 4B). The biodistribution of anti-mouse IC1-Cy5.5 in six normal organs was examined in comparison with IgG-Cy5.5 (Figure 4C). No preferential accumulation of anti-mouse IC1-Cy5.5 was observed in the examined organs (i.e., brain, lung, heart, liver, and kidney), but in the spleen. In addition, the interaction between the immune system and ICAM1 antibodies was studied by comparing the amounts of IC1-Cy5.5 and IgG-Cy5.5 taken up by circulating leukocytes in tumor-bearing BALB / c mice. IC1-Cy5.5 showed the same level of circulating leukocyte uptake as IgG-Cy5.5, suggesting that ICAM1 antibodies do not target normal leukocytes in the blood circulation (Figure 4D).

[0104] To evaluate the in vivo efficacy of IC1-MMAE and IC1-MMAF, a series of orthotopic TNBC models were assessed in standard-stage, late-stage, and refractory tumor stages. First, the in vivo efficacy of IC1-MMAE or IC1-MMAF in standard-stage orthotopic TNBC models (MDA-MB-436) was evaluated in tumors 100 mm 3Systemic treatment with IC1-MMAE or IC1-MMAF was examined by initiating tumor volume at a dose of 5 mg / kg when tumors reached a volume of 100 μg / kg (Figure 5A). Separate cohorts of tumor-bearing mice were treated with PBS (sham), ICAM1 antibody alone (IC1 Ab), or doxorubicin (free Dox) at equivalent dosages as controls. Both IC1-MMAE and IC1-MMAF elicited significant and sustained tumor regression in all animals tested throughout the duration of the experiment (Figures 5B and 5C). The antitumor activity of IC1-MMAE and IC1-MMAF was determined by measuring tumor weight, which was found to be 99.5% and 99.0% lower than that of the PBS-treated group, respectively. More importantly, IC1-MMAE and IC1-MMAF successfully eradicated MDA-MB-436 tumors in 70% (7 / 10) and 50% (4 / 8) of animals, respectively, without tumor recurrence over a 28-day period. In comparison, free Dox showed very limited efficacy against MDA-MB-436 tumors, while, as previously reported by others, 25~27 IC1 Ab demonstrated moderate antitumor efficacy, most likely by neutralizing the ICAM1 signaling cascade in MDA-MB-436 tumors. However, IC1-Ab treatment was significantly less effective than IC1-MMAE and IC1-MMAF treatments.

[0105] To demonstrate the potential of IC1-MMAE and IC1-MMAF in combating even more aggressive TNBC tumors, the in vivo efficacy of IC1-MMAE and IC1-MMAF was evaluated in the same orthotopic TNBC model (MDA-MB-436) at late stage. At late stage, MDA-MB-436 tumors were inoculated with 500 mm 3The tumors were able to grow to a volume of 1000 x 1000 mm (Figure 5A). IC1-MMAE significantly inhibited late-stage MDA-MB-436 tumor growth by 93.5% in all cases compared to the PBS-treated control group (Figure 5D). Although IC1-MMAF achieved 47.8% inhibition of tumor growth, this was significantly less effective than IC1-MMAE. Moreover, IC1-MMAE also eradicated late-stage MDA-MB-436 tumors in 20% (1 / 5) of animals, whereas IC1-MMAF did not eradicate the tumors. No weight loss was observed in animals treated with either IC1-MMAF or IC1-MMAE at both standard and late stages (Figures 5C and 5D). This suggests that these ICAM1 ADCs were well tolerated in TNBC-bearing nude mice at a dosage of 5 mg / kg.

[0106] Subsequently, the in vivo efficacy of IC1-MMAE and IC1-MMAF was evaluated in refractory (ADC-resistant) TNBC tumors. In this study, the effect of ICAM1 ADC on refractory TNBC tumors was examined by using ADC-resistant MDA-MB-231 cells previously established during the in vitro ADC efficacy study described previously. IC1-MMAE and IC1-MMAF were administered systemically at the same dosage of 5 mg / kg (Figure 6A). Both IC1-MMAE and IC1-MMAF potently attenuated refractory MDA-MB-231 tumor growth by 96.7% and 84.3%, respectively, in comparison with the PBS-treated control group (Figures 6B and 6C). IC1-MMAE also eradicated refractory tumors in 50% (3 / 6) of animals, whereas IC1-MMAF did not eradicate tumors. Similarly, no weight loss was observed during treatment of IC1-MMAE and IC1-MMAF at a dose of 5 mg / kg in this refractory TNBC model. These data demonstrate that both IC1-MMAE and IC1-MMAF are effective in treating orthotopic TNBC tumors in the standard phase of targeted therapy, however, IC1-MMAE is significantly more effective than IC1-MMAF in treating late-stage and refractory TNBC tumors. The higher efficacy of IC1-MMAE is likely due to its protease-cleavable MC-Vc-Pab linker, which mediates a faster mechanism of action than IC1-MMAF with a non-cleavable linker. Based on these in vivo efficacy results, IC1-MMAE was identified as forming an optimal ICAM1 ADC for TNBC treatment.

[0107] Next, the in vivo efficacy of IC1-MMAE was evaluated in a dose-dependent manner to determine the optimal dosage of IC1-MMAE for TNBC treatment. Three dosages of IC1-MMAE (1, 5, or 10 mg / kg) were tested along with a PBS control in a standard phase orthotopic TNBC model (MDA-MB-436) (Figure 6D). The antitumor activity of IC1-MMAE at 1, 5, and 10 mg / kg was determined to be 50.3%, 96.8%, and 94.5%, respectively, when compared to the PBS-treated control group (Figures 6E and 6F). The tumor eradication rates of IC1-MMAE at 1, 5, and 10 mg / kg were determined to be 0% (0 / 5), 40% (2 / 5), and 60% (3 / 5), respectively. These results suggest that IC1-MMAE administered at doses of 5 mg / kg or higher can induce significant and durable regression and eradication of TNBC tumors in vivo. No weight loss was observed as a result of IC1-MMAE at all doses tested. Therefore, 5 mg / kg was selected as the optimal dose for IC1-MMAE treatment in preclinical animal models.

[0108] Any potential in vivo toxicity of IC1-MMAE at 1, 5, and 10 mg / kg dosages was also evaluated using standard blood chemistry analysis. After 24 days, blood samples from each group were collected via cardiac puncture, and serum from the blood samples was examined for levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), two established serum biomarkers of hepatotoxicity. Even at the highest drug dosage (10 mg / kg), no elevation in AST or ALT levels was observed compared to the PBS group (Figure 6G). Similarly, nephrotoxicity of IC1-MMAE was assessed by measuring creatinine and blood urea nitrogen (BUN) levels. No nephrotoxicity was observed among the IC1-MMAE dosage groups studied (Figure 6G).

[0109] Finally, the maximum tolerated dose (MTD) of IC1-MMAE in healthy BALB / c mice was determined by intravenously administering IC1-MMAE at doses of 25, 50, or 75 mg / kg, approximately 5-15 times higher than the IC1-MMAE dose (5 mg / kg) used in the in vivo efficacy study (Figure 7A). No weight loss was observed at all IC-MMAE doses tested, even at the highest dose of 75 mg / kg (Figure 7B). Together, these in vivo toxicity studies strongly support the conclusion that IC1-MMAE is not only effective but also well tolerated in treating TNBC in preclinical in vivo models.

[0110] In addition to TNBC, the antitumor activity of four ICAM1 ADCs (ICAM1-DM4, ICAM1-DM1, ICAM1-MMAE, and ICAM1-MMAF) was tested against four additional human cancers that frequently express high levels of ICAM1 (Figure 8). The ICAM1 ADCs were observed to be effective in eradicating each of these additional ICAM1-expressing human cancer cell lines, including prostate, ovarian, skin, and lung cancer. These results indicate that the ICAM1 ADCs have the potential to be widely used as precision therapeutics for the treatment of other human cancers that express significant levels of the ICAM1 antigen.

[0111] Taken together, these results provide the first experimental evidence of utilizing ICAM1 as an effective ADC target for TNBC. Due to the high level of ICAM1 expression on TNBC cells and the low level of ICAM1 expression on healthy cells, ICAM1 ADCs can be used to specifically target cytotoxic drugs to TNBC cells while having minimal (if any) effect on non-cancer cells, as demonstrated. Alternative linkers and conjugated drugs for ICAM1 ADCs were also evaluated as described herein. While multiple ADCs were found to be effective in treating TNBC, ADCs with protease-cleavable linkers and microtubule inhibitors were found to be the formulation of choice for TNBC treatment among those tested due to their high and consistent efficacy in complete and sustained TNBC tumor regression and eradication. The in vivo efficacy of this ADC (IC1-MMAE) also significantly outperformed the clinically approved drug sacituzumab govitecan for treating refractory tumors in in vivo models of TNBC, likely as a result of synergy between its higher TNBC specificity and a better optimized ADC formulation. 35 The development of such an effective and well-tolerated therapeutic agent holds the promise of addressing the current unmet need in the treatment of TNBC.

[0112] material and method material Phycoerythrin (PE)-conjugated anti-human ICAM1 antibody (clone: ​​HCD54), PE-conjugated mouse IgG isotype (PE-IgG), anti-mouse ICAM1 antibody (clone #YN1 / 1.7.4), and RBC lysis buffer were purchased from BioLegend (San Diego, CA, USA). PE-conjugated anti-human TROP-2 antibody (clone #77220), mouse and rat IgG isotype controls were purchased from R&D systems (Minneapolis, MN, USA). Purified anti-human CD54 antibody (clone: ​​R6.5), MC-VC-PAB-MMAE, MC-MMAF, Mal-EBE-MaL-DM1, Mal-EBE-Mal-DM4 were obtained from MabPlex (Yantai, China). Lab-Tek II Chamber Slide System was obtained from Thermo Fisher Scientific. Doxorubicin, bovine serum albumin (BSA), AST activity assay kit, ALT activity assay kit, creatinine activity assay kit, and urea activity assay kit were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's PBS, Dulbecco's PBS, 4',6-diamidino-2-phenylindole (DAPI), Gibco Dulbecco's modified Eagle's medium (DMEM), Gibco DMEM / F12 (1:1), and Roswell Park Memorial Institute (RPMI)-1640 medium were purchased from Invitrogen (Carlsbad, CA). Dojindo cell counting kit CCK-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA).

[0113] cell culture Four human TNBC cell lines (MDA-MB-436, MDA-MB-468, MDA-MB-157, and MDA-MB-231), one mouse TNBC cell line (4T1), non-neoplastic human breast MCF10A cells, and human embryonic kidney HEK293 cells were purchased from American Type Culture Collection (Manassas, VA, USA). MDA-MB-436, MDA-MB-468, MDA-MB-157, MDA-MB-231, and HEK293 cells were cultured in DMEM, 4T1 cells were cultured in RPMI-1640, and MCF10A cells were cultured in DMEM / F12 (1:1). All media contained the recommended nutritional supplements. All cells were maintained in a humidified incubator at 37°C with 5% CO2.

[0114] Genomic analysis of ICAM1 expression in TNBC patients ICAM1 mRNA expression in human breast tumors and normal breast tissues was analyzed using the R2: Genomics Analysis and Visualization Platform database (hgserver1.amc.nl / ). The following four data sheets were used in the genomic analysis: Tumor Breast Invasive Carcinoma-TCGA-1097; Tumor Breast (TNBC)-Brown-198; Tumor Breast compendium- Halfwerk-947; Tumor Breast (mutation status)-Meijers-Heijboer-155. Detailed information of the microarray experiments and clinical samples is publicly available in the R2: Genomics Analysis and Visualization Platform database.

[0115] Flow cytometry 1×10 6The cells were harvested and rinsed twice with PBS. The obtained cells were blocked with 1% BSA in PBS in an ice bath for 30 minutes. After BSA blocking, the cells were incubated with PE-conjugated ICAM1 or TROP2 antibodies, respectively, for 1 hour at room temperature. Non-targeting PE-conjugated IgG was used as a control. The cells were rinsed three times with 1% BSA in PBS, suspended in PBS, and evaluated using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA).

[0116] Immunofluorescence staining 2×10 4 Cells were seeded in Lab-Tek II Chamber Slide System and cultured overnight at 37°C with 2mL cell culture medium. After removing the medium, cells were rinsed twice with PBS and fixed with 4% formaldehyde in PBS at rt for 10 min, followed by washing with PBS. Samples were blocked with 1% BSA in PBS for 30 min in an ice bath. After BSA blocking, samples were co-stained with PE-conjugated ICAM1 or TROP2 antibodies for 1 h and rinsed with PBS. DAPI was used to stain cell nuclei. Immunofluorescence staining samples were dried overnight in the dark and then examined using a Leica TCS SP5 confocal fluorescence microscope (Leica Microsystems, Buffalo Grove, IL, USA).

[0117] Imaging flow cytometry 1×10 6 Cells were seeded into each well of a 6-well chamber slide and allowed to adhere overnight. The adherent cells were then incubated with PE-conjugated ICAM1 antibody in 2 mL cell culture medium with 1% FBS at 37°C for 1 h. The cell monolayer was then harvested, rinsed twice with cold PBS, and resuspended. The cellular internalization rate of ICAM1 antibody in the treated cells was assessed using an Amnis imagestreamX Mark II imaging flow cytometer (Luminex, Austin, TX, USA).

[0118] In vitro binding and internalization of ICAM-1 antibodies The specific binding of ICAM1 antibodies to human TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-157) in vitro was assessed using phycoerythrin (PE)-ICAM1 antibodies. Non-cancerous MCF10A cells were used as a control. Cells were cultured at 5 × 10 in 8-well chamber slides. 3 Cells were seeded at a density of 1000 cells / well. After 24 h recovery, the entire medium was replaced with PE-ICAM1 antibody with 1% FBS. Cells were incubated in PE-ICAM1-containing medium for an additional 4 h at 37°C. Cell monolayers were then rinsed with cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS solution. Cell nuclei were counterstained with 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) using ProLong Gold Antifade Mountant. Fluorescent images were acquired and analyzed using a Zeiss LSM 880 confocal microscope (Oberkochen, Germany).

[0119] Preparation and characterization of ICAM1 ADCs ICAM1-DM1, ICAM1-DM4, ICAM1-MMAE, and ICAM1-MMAF ADCs were prepared by conjugating ICAM1 antibody with Mal-EBE-Mal-DM1, Mal-EBE-Mal-DM4, MC-VC-PAB-MMAE, or MC-MMAF, respectively. The drug-antibody ratios (DARs) of the synthesized ADCs were characterized by hydrophobic interaction chromatography.

[0120] In vitro potency and cytotoxicity assays Human TNBC cell lines and control cells were plated in 96-well plates at 5 × 10 3Cells were seeded at a density of 1000 cells / well and allowed to adhere overnight. The culture medium was then replaced with medium containing IgG or ICAM1 conjugated to DM1, DM4, MMAE, or MMAF at various drug concentrations. After culturing the cells for another 48 hours, cytotoxicity was determined by CCK-8 assay following the protocol provided by the supplier. After removing the drug-containing medium, the cells were carefully rinsed with PBS, followed by the addition of CCK-8-containing medium solution. The cells were then incubated in CCK-8 medium for 4 hours. The plates were read at an absorbance wavelength of 450 nm using a Molecular Devices SpectraMax microplate reader (San Jose, CA, USA). Cell viability was determined by comparing the absorbance of cells incubated with the drug to that of control cells incubated in the absence of drug.

[0121] Orthotopic TNBC Mouse Model The mouse studies presented in this study were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children's Hospital. For tumor specificity and in vivo biodistribution studies, 2 × 10 6 Orthotopic TNBC were implanted by injecting TNBC cells (MDA-MB-436 or 4T1) into the fourth right mammary fat pad of 6- to 8-week-old female nude or BALB / c mice (Charles River, Wilmington, MA, USA). Tumors were grown until their volume reached at least 200–300 mm. 3Tumor-bearing mice were allowed to grow for 2-3 weeks until they were mature, then randomly divided into various treatment groups (n>5 per group) and received intravenous injections of IgG-Cy5.5, IC1-Cy5.5, or IC1-MMAE-Cy5.5 at a dosage of 5 mg / kg mouse weight. 48 hours after injection, in vivo NIR fluorescence imaging was performed on treated animals using an IVIS Lumina II system (Caliper, Hopkinton, MA, USA). Mice were then euthanized by CO2 and 500 μL mouse blood was immediately collected via cardiac puncture. NIR fluorescence intensity of various organs (brain, heart, liver, lungs, kidneys, and spleen) and excised tumors was measured using an IVIS Lumina II. Mouse white blood cells were isolated from whole blood via centrifugation at 1200 rpm for 15 min, followed by removal of red blood cells using RBC lysis buffer. The fluorescence intensity of IgG-Cy5.5 or IC1-Cy5.5 taken up by leukocytes was quantified using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA).

[0122] For in vivo efficacy studies, orthotopic TNBC tumors (MDA-MB-436 or MDA-MB-231) were established in 6-8 week old female nude mice as described above. Tumors were grown to 100 mm 3 or 500mm for late stage procedures 3Tumor-bearing mice were then randomly assigned to different treatment groups (n>5 per group) and received treatment with PBS (sham), free Dox, IC1 Ab, IC1-MMAE, or IC1-MMAF at equivalent doses of 5mg / kg per week via tail vein injection for 3 weeks. Tumor growth was monitored weekly using calipers. At the endpoint, animals were euthanized with CO2, and orthotopic tumors were excised and their masses were measured. In a dose-dependent experiment, three increasing doses of IC1-MMAE (1, 5, and 10mg / kg) were evaluated using the same protocol in orthotopic TNBC tumors (MDA-MB-436). Mice were then euthanized with CO2, and 500μL mouse blood was immediately collected via cardiac puncture. The collected blood was incubated at room temperature for 20 minutes to allow coagulation, and then mouse serum was isolated after centrifugation at 2,000×g for 10 minutes in a refrigerated centrifuge. Serum levels of ALT, AST, creatinine, and BUN were determined using their activity assay kits purchased from Sigma-Aldrich (St. Louis, MO, USA) with the provided protocols. In an MTD study, the in vivo tolerability of IC1-MMAE was examined in 6-8 week-old female BALB / c mice. Healthy mice were randomly divided into various treatment groups (n=10 per group) and received intravenous injections of IC1-MMAE at three increasing dosages (25, 50, and 75 mg / kg) per injection. After injection, mouse weight loss was used as an acute toxicity index and was closely monitored for up to 14 days.

[0123] statistical analysis All experimental data were obtained in triplicate and are presented as mean ± SD unless otherwise stated. One- and two-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used to analyze statistical variance when making multiple comparisons. All statistical analyses were performed using OriginPro 8 software.

[0124] Examples of linker and drug structures in ADCs The structures of the linkers and drugs used in this disclosure are provided below. Name: Mal-EBE-Mal-DM1 Chemical structure:

change

[0125] Name: Mal-EBE-Mal-DM4 Chemical structure:

change

[0126] Name: MC-VC-PAB-MMAE Chemical structure:

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[0127] Name: MC-MMAF Chemical structure:

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[0128] References

Table 3

[0129]

Table 4

[0130]

Table 5

[0131]

Table 6

[0132] [Table 7]

[0133] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0134] Articles such as "a," "an," and "the" may mean one or more than one, unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between two or more members of a group is deemed to satisfy the case where one, more than one, or all of the members of the group are present, unless indicated to the contrary or otherwise clear from the context. The present disclosure of groups including "or" between two or more members of the group provides embodiments where exactly one member of the group is present, embodiments where more than one member of the group is present, and embodiments where all members of the group are present. For the sake of brevity, the embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0135] It should be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or from one or more relevant recitations are incorporated into another claim. For example, a claim (dependent on another claim) may be modified to include one or more limitations found in any other claim dependent on the same base claim. Furthermore, if a claim describes a composition, it should be understood that a method of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art (if any) is also included, unless otherwise indicated or unless it is obvious to one of ordinary skill in the art that an inconsistency or inconsistency would occur.

[0136] Where elements are presented as a list (for example, in the form of a Markush group), it is to be understood that any possible subgroups of the elements are also disclosed, and that any element or any subgroup of elements can be removed from the group. It should also be noted that the term "comprising" is intended to be open, allowing for the inclusion of additional elements or steps. In general, where an embodiment, product, or method is referred to as comprising specific elements, features, or steps, it should be understood that an embodiment, product, or method consisting of or consisting essentially of such elements, features, or steps is also provided. For the sake of brevity, the embodiments are not individually described in detail herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0137] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any particular value within the stated range, in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For the sake of brevity, the values ​​in each range are not specifically described individually herein, but it will be understood that each of these values ​​is provided herein and may be specifically claimed or disclaimed. Unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, it should also be understood that values ​​expressed as ranges can assume any subrange within the given range (wherein the endpoint of the subrange is expressed with the same precision as the tenth of the unit of the lower limit of the range).

[0138] Where web sites are provided, URL addresses are provided as browser-inoperable code and the periods in each web address are indicated in parentheses. The actual web addresses do not contain parentheses.

[0139] In addition, it should be understood that any specific embodiment of the present disclosure may be expressly excluded from any one or more claims. Where a range is given, any value within the range may be expressly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more claims. For the sake of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are not expressly set forth herein.

Claims

1. A composition for use in a method for treating intercellular adhesion molecule 1 (ICAM1)-expressing cancer, the composition comprising an effective amount of an antibody-drug conjugate (ADC) comprising an ICAM1 antibody conjugated to a drug, the method comprising administering the composition to a subject in need thereof.

2. 2. The composition of claim 1, wherein the drug is selected from the group consisting of: N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)mertansine (DM1), N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF).

3. The composition of claim 2, wherein the drug is MMAE.

4. The composition of claim 2, wherein the drug is MMAF.

5. The composition according to any one of claims 1 to 4, wherein the ICAM1 antibody and the drug are conjugated via a linker.

6. The composition of claim 5 , wherein the linker is a cleavable linker.

7. 7. The composition of claim 6, wherein the cleavable linker is selected from the group consisting of N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), Sulfo-SPDB, valine-citrulline (Val-cit), acetylbutyrate, CL2A, and maleimidocaproyl (MC), and Mal-EBE-Mal.

8. The composition of claim 5 , wherein the linker is a non-cleavable linker.

9. 9. The composition of claim 8, wherein the non-cleavable linker is selected from the group consisting of: N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), MC-VC-PAB.

10. The composition of any one of claims 1 to 9, wherein the ICAM1 antibody is selected from the group consisting of an IgG, an Ig monomer, a Fab fragment, a F(ab')2 fragment, an Fd fragment, a scFv, a scAb, a dAb, an Fv, an affibody, a bispecific antibody, a single domain heavy chain antibody, and a single domain light chain antibody.

11. The composition according to any one of claims 1 to 10, wherein the ICAM1 antibody is a chimeric or humanized antibody.

12. The composition of any one of claims 1 to 11, wherein the ICAM1 antibody is R6.5 or HCD54.

13. The composition of claim 12, wherein the ICAM1 antibody is a chimeric or humanized form of R6.5 or HCD54.

14. The composition of any one of claims 1 to 13, wherein the ratio of ICAM1 antibody to drug in the ADC is 1:1 to 1:

10.

15. The composition of claim 14, wherein the ratio of ICAM1 antibody to drug in the ADC is 1:

4.

16. The composition of any one of claims 1 to 15, wherein the composition is administered via injection.

17. The composition of claim 16, wherein the injection is an intravenous injection or an intratumoral injection.

18. The composition of any one of claims 1 to 17, wherein the composition is administered via intravenous injection.

19. The composition of any one of claims 1 to 18, wherein the ADC is administered at a dosage of 1 mg / kg to 75 mg / kg.

20. 20. The composition of claim 19, wherein the ADC is administered at a dosage of 5 mg / kg.

21. The composition of any one of claims 1 to 20, wherein the composition is administered at a frequency ranging from once a week to once every other month.

22. The composition according to any one of claims 1 to 21, wherein the subject is a human.

23. The composition of any one of claims 1 to 22, wherein the ICAM1-expressing cancer is breast cancer, prostate cancer, ovarian cancer, melanoma, or lung cancer.

24. 24. The composition of claim 23, wherein the breast cancer is triple-negative breast cancer (TNBC).

25. 1. A composition for use in a method for treating triple-negative breast cancer (TNBC), the composition comprising an effective amount of an antibody-drug conjugate (ADC) comprising a chimeric intercellular adhesion molecule 1 (ICAM1) antibody conjugated to monomethylauristatin E (MMAE) via an MC-VC-PAB linker, the method comprising administering the composition to a subject in need thereof.

26. 1. A composition for use in a method of treating triple-negative breast cancer (TNBC), the composition comprising an effective amount of an antibody-drug conjugate (ADC) comprising a chimeric intercellular adhesion molecule 1 (ICAM1) antibody conjugated to monomethylauristatin F (MMAF) via an MC linker, the method comprising administering the composition to a subject in need thereof.

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