Intercellular adhesion molecule 1 (ICAM1) antibody drug conjugate and uses thereof

Abstract

To provide methods to improve pancreatic cancer treatment and stratify patient populations for precision medicine.SOLUTION: The present invention provides a method of treating pancreatic cancer, the method 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.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 891,170, entitled "Intercellular Adhesion Molecule 1 (ICAM1) Antibody-Drug Conjugates and Their Use," filed on August 23, 2019, the entire content of which is incorporated herein by reference.

Background Art

[0002] Background Pancreatic cancer (PC) remains one of the most lethal diseases, accounting for 56,770 deaths in the United States in 2019, corresponding to 7% of all cancer mortalities. Despite the increasing research on immunotherapy and nanomedicine for treatment in recent years, the prognosis for PC patients is extremely poor, with less than 8% 5-year survival.

Summary of the Invention

[0003] Summary The present disclosure is based, at least in part, on the surprising finding that intercellular adhesion molecule 1 (ICAM1) can be targeted to improve the treatment of pancreatic cancer and to stratify patient populations towards precision medicine. The immunosuppressive microenvironment of pancreatic cancer tumors presents several challenges for effective treatment. For example, the tumor microenvironment of pancreatic cancer tumors is often characterized by a desmoplastic stroma and poor vascularization, which create physical barriers that prevent T cells or drugs from efficiently penetrating the tumor. These limitations are, at least in part, addressed by the present disclosure.

[0004] Provided herein are, in some aspects, antibody-drug conjugates (ADCs) comprising intercellular adhesion molecule 1 (ICAM1) that are useful in the treatment of pancreatic cancer. As described below, the use of ADCs comprising ICAM1 antibodies enables preferential targeting of pancreatic cancer cells over non-cancerous cells, which can improve the therapeutic concentration range of the drug and limit toxicity. Given the high genetic heterogeneity of pancreatic cancer, it is also challenging to predict treatment sensitivity among patient populations. Consequently, a further aspect of the present disclosure provides a method of identifying a patient population for treatment with an ICAM1 antibody or an ADC comprising an ICAM1 antibody in a subject with pancreatic cancer.

[0005] Aspects of the present disclosure provide a method of treating pancreatic cancer 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.

[0006] In some embodiments, the drug is selected from the group consisting of N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin. In some embodiments, the drug is DM1. In some embodiments, the ICAM1 antibody and the drug are conjugated via a linker.

[0007] 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), acetyl butyrate, and CL2A. In some embodiments, the cleavable linker is Val-cit.

[0008] 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). In some embodiments, the non-cleavable linker is an N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

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

[0010] In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is from 1:1 to 1:10. In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is 1:4. In some embodiments, the ADC is administered via injection. In some embodiments, the injection is intravenous injection or intratumoral injection.

[0011] A further aspect of the present disclosure provides a method of treating pancreatic cancer, 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 N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1) via an N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

[0012] A further aspect of the present disclosure provides a method of predicting responsiveness to treatment with an antibody-drug conjugate (ADC) comprising an ICAM1 antibody or an ICAM1 antibody conjugated to a drug in a subject having pancreatic cancer, the method comprising: (i) administering to the subject an effective amount of an ICAM1 antibody labeled with an imaging agent; and (ii) visualizing the tumor via imaging; (iii) determining the level of ICAM1 on the tumor, wherein a higher level of ICAM1 indicates that the subject will respond better to treatment with the ICAM1 antibody or ADC as compared to a subject having a lower level of ICAM1 (e.g., identifying a subject as responding better to treatment when the level of ICAM1 is higher as compared to a subject having a tumor with a lower level of ICAM1).

[0013] In some embodiments, the ICAM1 antibody in (i) is labeled with DTPA-Gd. In some embodiments, the visualization in (ii) is via magnetic resonance imaging (MRI). In some embodiments, the method further comprises administering to the subject an effective amount of the ICAM1 antibody or ADC, who is predicted to respond to the treatment for treating pancreatic cancer.

[0014] In some embodiments, the drug is selected from the group consisting of N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) mertansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin. In some embodiments, the drug is DM1. In some embodiments, the ICAM1 antibody and the drug are conjugated via a linker.

[0015] 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), acetyl butyrate, and CL2A. In some embodiments, the cleavable linker is Val-cit.

[0016] 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 maleimidomethyl cyclohexane-1-carboxylate (MCC). In some embodiments, the non-cleavable linker is the N-succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker.

[0017] In some embodiments, the ICAM1 antibody is selected from the group consisting of IgG, Ig monomer, Fab fragment, F(ab')2 fragment, Fd fragment, scFv, scAb, dAb, Fv, affibody, bispecific antibody, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, the ICAM1 antibody is enlimomab or HCD54.

[0018] In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is from 1:1 to 1:10. In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is 1:4. A further aspect of the present disclosure provides an antibody-drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to a drug.

[0019] In some embodiments, the drug is selected from the group consisting of: N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) mertansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin. In some embodiments, the drug is DM1. In some embodiments, the ICAM1 antibody and the drug are conjugated via a linker.

[0020] 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), acetyl butyrate, and CL2A. In some embodiments, the cleavable linker is Val-cit.

[0021] 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 maleimidomethyl cyclohexane-1-carboxylate (MCC). In some embodiments, the non-cleavable linker is N-succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker.

[0022] In some embodiments, the ICAM1 antibody is selected from the group consisting of: IgG, Ig monomer, Fab fragment, F(ab')2 fragment, Fd fragment, scFv, scAb, dAb, Fv, affibody, bispecific antibody, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, the ICAM1 antibody is enlimomab or HCD54.

[0023] In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is from 1:1 to 1:10. In some embodiments, the ratio of the ICAM1 antibody to the drug in the ADC is 1:4.

[0024] A further aspect of the present disclosure provides an antibody-drug conjugate (ADC) comprising an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) mertansine (DM1) via an N-succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Brief Description of the Drawings The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or substantially identical component that is described in various figures is represented by like numerals. For the sake of clarity, not all components may be shown in all of the figures. In the drawings:

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DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed description of one embodiment To date, pancreatic cancer (PC) remains one of the most lethal diseases, accounting for 56,770 deaths in the United States in 2019, corresponding to 7% of all cancer mortalities. Despite the recent increase in research on immunotherapy and nanomedicine-based treatments, the prognosis for PC patients remains extremely poor, with a 5-year survival of less than 8%. These unfavorable outcomes are largely due to the immunosuppressive tumor microenvironment (TME) of PC tumors, which is characterized by a fibrotic stroma and insufficient angiogenesis. Such a TME creates a physical barrier that prevents T cells or nanomedicine from efficiently infiltrating the tumor and directly interacting with PC cells, leading to undesirable effects. Developing novel targeted therapeutic agents that can improve infiltrated PC tumors while maintaining potent tumor-specific effects highlights an essential need.

[0032] Antibody-drug conjugates (ADCs) have shown promising clinical efficacy against several cancers, including aggressive solid tumors such as breast cancer with insufficient response to T cell immunotherapy. Although several PC-targeted ADCs utilizing conventional PC targets (e.g., EGFR, EpHA2, and mesothelin) have been developed, there is still a lack of systematic and quantitative comparison of established PC targets and other candidates at the level of their cell surface proteins.

[0033] Provided herein is, in some aspects, a fair and quantitative screening of cell surface proteins to discover more optimal PC immunotherapy targets and facilitate the development of PC-targeted ADCs. ICAM1 has been identified as a potential PC immunotherapy target. The ICAM1 ADCs described herein induced potent and durable PC tumor regression in vivo. The present disclosure also contemplates the use of ICAM1 as a target for immunotherapy for pancreatic cancer (including, without limitation, T cell-based immunotherapies such as CART and checkpoint blockade).

[0034] Furthermore, the present disclosure provides a non-invasive MRI approach to identify ICAM1-expressing tumors suitable for ICAM1-targeted immunotherapy. Such patients are, in some embodiments, administered an ICAM1 ADC for the treatment of PC.

[0035] Aspects of the present disclosure provide an antibody-drug conjugate (ADC) comprising intercellular adhesion molecule 1 (ICAM1) and a drug, methods of using the conjugate in the treatment of pancreatic cancer, and methods of predicting responsiveness to treatment with an ICAM1 antibody or an ICAM1 antibody-drug conjugate (ADC).

[0036] Antibody-drug conjugate (ADC) 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 that express ICAM1. ICAM1 is a cell surface glycoprotein that has been shown to bind to integrins of the type CD11a / CD18 or CD11b / CD18 and is involved in mediating cell-cell interactions and promoting leukocyte endothelial transmigration. ICAM1 is also referred to as ICAM-1, BB2, surface antigen cluster 54 (CD54), and P3.58.

[0037] 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: MASTRAKPTLPLLLALVTVVIPGPGDAQVSIHPREAFLPQGGSVQVNCSSSCKEDLSLGLETQWLKDELESGPNWKLFELSEIGEDSSPLCFENCGTVQSSASATITVYSFPESVELRPLPAWQQVGKDLTLRCHVDGGAPRTQLSAVLLRGEEILSRQPVGGHPKDPKEITFTVLASRGDHGANFSCRTELDLRPQGLALFSNVSEARSLRTFDLPATIPKLDTPDLLEVGTQQKLFCSLEGLFPASEARIYLELGGQMPTQESTNSSDSVSATALVEVTEEFDRTLPLRCVLELADQILETQRTLTVYNFSAPVLTLSQLEVSEGSQVTVKCEAHSGSKVVLLSGVEPRPPTPQVQFTLNASSEDHKRSFFCSAALEVAGKFLFKNQTLELHVLYGPRLDETDCLGNWTWQEGSQQTLKCQAWGNPSPKMTCRRKADGALLPIGVVKSVKQEMNGTYVCHAFSSHGNVTRNVYLTVLYHSQNNWTIIILVPVLLVIVGLVMAASYVYNRQRKIRIYKLQKAQEEAIKLKGQAPPP (SEQ ID NO: 1).

[0038] The UniProtKB accession number P05362 encodes ICAM1 from Homo Sapiens and has the following sequence: MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPP (SEQ ID NO: 2).

[0039] 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.

[0040] The antibodies of the present disclosure are 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.

[0041] Non-limiting examples of ICAM1 antibodies include clone HCD54 (commercially available "HCD54" from BioLegend, catalog # 322702), UV3, RR1.1, R6.5 (commercially available BIRR-1 or enlimomab from Thermo Fisher Scientific, catalog # BMS1011), and BI-505. R6.5 (enlimomab) is a monoclonal mouse antibody produced by ATCC HB-9580 hybridoma cells as described, for example, in U.S. Patent No. 5,324,510, which is incorporated herein by reference.

[0042] UV3 is a monoclonal antibody and has been shown to bind to ICAM-1 on myeloma cells. In some embodiments, the ICAM1 antibody is the 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, which are each incorporated herein by reference. RR1.1 is a monoclonal ICAM1 antibody. See, for example, Rothlein and Springer, 1986 J.Exp.Med. 163, 1132-1149, which is incorporated herein by reference. HCD54 is a monoclonal ICAM1 antibody. BI-505 is a fully human ICAM1 monoclonal antibody. See, for example, Hansson et al., Clin Cancer Res. 2015 Jun 15;21(12):2730-6, which is incorporated herein by reference.

[0043] The term "bind" refers to the association of two entities (e.g., two proteins). Two entities (e.g., two proteins) bind to each other when the affinity (KD) between them is < 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. One of ordinary skill in the art is familiar with ways to assess the affinity of two entities (e.g., two proteins).

[0044] The term "antibody" encompasses whole antibodies (immunoglobulins having two heavy chains and two light chains), antibody mimetics, and antibody fragments. "Immunoglobulin (Ig)" is a large Y-shaped protein mainly produced by plasma cells 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 fragment" encompasses any antigen-binding fragment (i.e., "antigen-binding site") 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 its antigen-binding site. 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 region and the VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each of VH and VL is composed of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody may also 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 of the classical complement system (C1q). In some embodiments, the antibody is an immunoglobulin (Ig) monomer. The antibody may be a polyclonal antibody or a monoclonal antibody.

[0045] In some embodiments, an antibody is a glycoprotein heterotetramer composed of two identical L chains and two H chains (IgM antibodies consist of five basic heterotetramer units and an additional polypeptide called the J chain and thus contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 of the basic four-chain units and the J chain). For IgG, the four-chain unit generally weighs 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 of the H and L chains also has regularly spaced intra-chain disulfide bridges. Each H chain has at its N-terminus 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 at its N-terminus a variable domain (VL), followed by a constant region (CL) at its other end. VL is aligned with VH, and CL is aligned with the first constant region of the heavy chain (CH1). Certain amino acid residues are thought to form the interface between the variable domains of the light and heavy chains. Pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of non-limiting examples of various classes of antibodies, see, by way of 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 IgG.

[0046] The L chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequence of their heavy chain constant regions (CH), immunoglobulins can be assigned to various classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have 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 the sequence and function of the CH. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0047] The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 - amino - acid span of the variable domain. Instead, the V region consists of relatively invariant stretches called framework regions (FRs) of 15 - 30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" each 9 - 12 amino acids long. The variable domains of native heavy and light chains each contain four FRs (most of which introduce a β - sheet conformation) connected by three hypervariable regions, where the FRs form loops connecting to the β - sheet structure and, in some cases, form part of the β - sheet structure. The hypervariable regions in each chain are held together in close proximity to the hypervariable regions from the other chain and to the FRs, contributing to the formation of the antigen - binding site of the antibody (see, for example, 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 do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the participation of the antibody in antibody - dependent cell - mediated cytotoxicity (ADCC).

[0048] In some embodiments, the antibody is a monoclonal antibody. A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the population of antibodies contains individual antibodies that are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain a variety of antibodies directed against various 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 can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any specific 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, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) (incorporated herein by reference).

[0049] The monoclonal antibodies described herein encompass "chimeric" antibodies and fragments of such antibodies (so long as they exhibit the desired biological activity), wherein a portion of the heavy and / or light chains in said antibody is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (see U.S. Patent 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 non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0050] 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 the blood, secretions, or other body fluids of a mammal, or from eggs. Polyclonal antibodies may also be recombinant. A recombinant polyclonal antibody is a polyclonal antibody produced by the use of recombinant techniques. Polyclonal antibodies produced recombinantly generally contain a high concentration of different antibody molecules, and all or most of these (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or more) exhibit the desired binding activity against an antigen composed of more than one epitope.

[0051] In some embodiments, the antibody is "humanized" for use in humans (e.g., as a therapeutic agent). A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains minimal sequence derived from the non-human antibody. A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient are replaced by residues from the hypervariable regions of a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) having the desired antibody specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops in the variable domains correspond to the hypervariable loops of the non-human immunoglobulin and all or substantially all of the FRs are the FRs of the human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region 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).

[0052] 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 the 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 carried out by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody are: (i) the Fab fragment, which is a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) the F(ab')2 fragment, which is a bivalent fragment that includes two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, which consists of the VH domain and the CH1 domain; (iv) the Fv fragment, which consists of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment, which consists of the VH domain (as described, for example, in Ward et al., (1989) Nature 341:544-546, which is incorporated herein by reference); and (vi) isolated complementarity-determining regions (CDRs). Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these can be joined using recombinant methods and a synthetic linker that allows them to form a monovalent molecule as a single protein chain that pairs the VL region and the VH region (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 can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0053] In some embodiments, the antibody fragment may be an Fc fragment, an Fv fragment, or a single-change Fv fragment. The Fc fragment includes the carboxy termini of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcRs) found on certain types of cells.

[0054] The Fv fragment is the minimal antibody fragment that contains the complete site for antigen recognition and binding. This fragment consists of a dimer of the variable region domains of one heavy chain and one light chain held together by non-covalent, tight binding. From these folds, two domains give rise to six hypervariable loops (three loops from each of the 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 containing 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.

[0055] The single-chain Fv, also abbreviated as "sFv" or "scFv", is an antibody fragment that includes the VH antibody domain and the VL antibody domain that, when connected, form a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains that allows the sFv to form the structure desired for antigen binding (e.g., as described in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, incorporated herein by reference). In some embodiments, the antibody is a dimeric scFV (bispecific antibody), a trimeric scFV (trispecific antibody), or a tetrameric scFV (tetraspecific antibody).

[0056] The antibodies of the present disclosure include antibody mimetics that include affibody molecules. An affibody is a small protein containing three helix bundles that function as antigen-binding molecules (e.g., antibody mimetics). Generally, an affibody is approximately 58 amino acids in length and has 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. Specific affibody molecules that bind to a desired target protein can be isolated from a pool (library) containing billions of different variants using methods such as phage display.

[0057] 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 (as opposed to the portion inside the cytosol) and on the cell surface. The extracellular portion of ICAM1 typically includes.

[0058] 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 isolating antibody-producing B lymphocytes from blood, bone marrow, lymph nodes, or spleen. Alternatively, antibody-producing cells may be isolated from an animal and exposed in vitro to the allergen (against which the antibody is to be raised). The antibody-producing cells may then optionally be cultured to obtain a population of antibody-producing cells, after fusion with an immortalized cell line such as a myeloma. In some embodiments, B lymphocytes as starting material may be isolated from the tissue of an allergic patient 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, transgenic mice or other animals with human immunoglobulin genes (as disclosed, for example, in U.S. Patent No. 5,939,598) may be immunized to stimulate the in vivo production of specific antibodies and antibody-producing cells prior to preparing polyclonal antibodies from the animals by extraction of B lymphocytes or purification of polyclonal serum.

[0059] Monoclonal antibodies are typically produced by cell culture involving the fusion of 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 parental cell on microtiter wells. The antibodies secreted by different clones are then assayed for their ability to bind to their antigen (in tests such as ELISA or antigen microarray assays) or immunoblotting. The most productive and stable clones are then selected for future use.

[0060] II. Drugs Drugs suitable for use in an ADC include agents having therapeutic activity against pancreatic cancer. 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 small molecule that inhibits cell proliferation.

[0061] Non-limiting examples of drugs suitable for use in an ADC include N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (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.

[0062] The term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or created artificially (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, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of the 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 certain embodiments, the molecular weight of the 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. Combinations of the above ranges (e.g., at least about 200 g / mol and about 500 g / mol or less) are also possible.

[0063] Any known chemotherapeutic agent may be used as the drug in the ADCs described herein. Exemplary chemotherapeutic agents include, but are not limited to: 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.

[0064] 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 the ICAM1 antibody to the drug in the ADC may be from 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 the ICAM1 antibody to the drug in the ADC is 1:4.

[0065] The ICAM1 antibody may be conjugated to the second entity either directly or via a linker. As used herein, "conjugated" or "attached" preferably means that two entities are linked with an affinity sufficient to achieve a therapeutic or diagnostic benefit of the linkage between the two entities. In some embodiments, the linker conjugates the ICAM1 antibody to the 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.

[0066] In some embodiments, the linker is a cleavable linker. As used herein, a cleavable linker is capable of releasing the 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 a pH of 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.

[0067] 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), acetyl butyrate, and CL2A. In some embodiments, the cleavable linker is Val-cit. Also see, for example, Donaghy, MAbs. 2016 May-Jun;8(4):659-71.

[0068] In some embodiments, the linker is non-cleavable. In some embodiments, a non-cleavable linker is a linker that is not cleaved in the systemic circulation in a subject. In some embodiments, a 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), and maleimidomethyl cyclohexane-1-carboxylate (MCC). In some embodiments, a non-cleavable linker is an N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker.

[0069] 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.

[0070] An ADC comprising an ICAM1 antibody conjugated to a drug may also be advantageously used in therapy, in part because the drug (e.g., a chemotherapeutic agent) 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%, at least 99% compared to the free form of the drug.

[0071] Other ICAM1 antibody conjugates Either the ICAM1 antibody and / or the ADCs of the present disclosure may be conjugated to an imaging agent that may also be useful for predicting treatment sensitivity in pancreatic cancer subjects. For example, imaging agents for computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and endoscopic detection (e.g., endoscopic ultrasound examination) may be used, and these may include contrast agents. See, for example, 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 contrast 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, for example, Carr et al., AJR Am J Roentgenol. 1984 Aug;143(2):215-24.

[0072] One or more imaging agents may be conjugated to the ICAM1 antibody or the ADCs 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 the ICAM1 antibody or ADC to the imaging agent may be from 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 the ICAM1 antibody or ADC to the imaging agent is 1:4. Any of the linkers disclosed herein may be used to conjugate the imaging agent to the ICAM1 antibody or the ADCs described herein. The imaging agent may be visualized by a suitable detection method (e.g., by CT, PET, MRI, ultrasound, and / or endoscopic detection).

[0073] Pharmaceutical Compositions and Their Use Compositions comprising any of the ADCs or other ICAM1 antibody conjugates disclosed herein are encompassed by the present disclosure. In some embodiments, the composition is formulated as a pharmaceutical composition for administration to a subject.

[0074] The subject may have pancreatic cancer, be suspected of having pancreatic cancer, or be at risk of pancreatic cancer. Pancreatic cancer is classified based on the cell type from which the tumor begins. The most common type of pancreatic cancer is pancreatic adenocarcinoma, which is cancer of the exocrine pancreas. In contrast, pancreatic neuroendocrine tumors (NETs) or islet cell tumors begin in neuroendocrine cells.

[0075] Pancreatic cancer may also be stratified based on whether the cancer has metastasized. Pancreatic cancer may be stage 0 (in situ cancer), stage I, stage II (e.g., stage IIA or stage IIB), stage III, or stage (IV). A non-limiting staging method is the TNM system that evaluates the extent of the tumor (T), spread to lymph nodes near the cancer (N), and whether the cancer has spread to distant sites (M). Then various levels of T, N, and M (e.g., Table 1) may be used to determine the stage of pancreatic cancer (e.g., Table 2). Tables 1-2 show the tumor classification of the pancreas as per 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.

[0076] Table 1. Non-limiting examples of TNM staging definitions [Table 1]

[0077] Table 2. Staging levels of the pancreas [Table 2]

[0078] In some embodiments, any of the pharmaceutical compositions disclosed herein that include an imaging agent, when administered to a subject in an effective amount, determines the level of ICAM1 in the tumor of a pancreatic cancer subject (by way of example, CT, PET, MRI, and endoscopic detection (by way of example, endoscopic ultrasound)). The imaging method for determining the level of ICAM1 described herein is advantageous as compared to conventional methods (by way of example, biopsy and analysis of tissue obtained from a biopsy). The imaging method (by way of example, MRI) is non-invasive and provides a comprehensive view of the tumor with respect to the ICAM1 level, thereby providing a more accurate assessment for predicting the outcome and / or responsiveness to tumor treatment (by way of example, treatment with an ICAM1 antibody or an ADC that includes an ICAM1 antibody).

[0079] In some embodiments, the level of ICAM1 is detected in a pancreatic cancer subject to whom a pharmaceutical composition of the present disclosure that includes an ICAM1 antibody and an imaging agent has been administered. 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 a control. In some embodiments, the level of ICAM1 detected in the subject's tumor is substantially the same as a control.

[0080] In some embodiments, the control is a subject having a tumor with a known level of ICAM1. In some embodiments, the control is the level of ICAM1 in the pancreas of a subject without a tumor. In some embodiments, the control is a subject having a tumor with a low level of ICAM1. In some embodiments, the low level of ICAM1 is undetectable. In some embodiments, the control is a subject having a tumor with 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 the pancreas of a healthy subject.

[0081] In some embodiments, the level of ICAM1 detected in a tumor using the methods disclosed herein suggests pancreatic cancer in a subject responsive to treatment with an antibody-drug conjugate (ADC) comprising an ICAM1 antibody or an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to a drug. In some embodiments, a higher level of ICAM1 detected in a tumor is identified as responsive to treatment with an ICAM1 antibody or an ADC disclosed herein as compared to tumors of subjects with lower levels of ICAM1. In some embodiments, a subject having a higher level of ICAM1 in a 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% more responsive to treatment with a composition comprising an ICAM1 antibody (such as an ICAM1 ADC and / or an ICAM1 antibody not conjugated to a drug) as compared to a subject having a lower level of ICAM1 in a tumor. In some embodiments, the methods disclosed herein include administering an ICAM1 antibody or an ADC antibody disclosed herein after identifying a subject as responsive.

[0082] In some embodiments, the level of ICAM1 detected in a tumor using the methods disclosed herein suggests the stage of pancreatic cancer. In some embodiments, the level of ICAM1 detected in a tumor suggests stage 0, stage I, stage II, stage III, or stage IV.

[0083] Without being bound by 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 pancreatic tumors and treating the tumors.

[0084] 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 tumor size 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.

[0085] 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% compared to 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.

[0086] In some embodiments, administration of an ICAM1 antibody and / or an ADC disclosed herein that includes 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.

[0087] In some embodiments, administration of an ICAM1 antibody and / or an ADC disclosed herein that includes an ICAM1 antibody, or a pharmaceutical composition thereof, does not reduce the viability of healthy cells. In some embodiments, administration of an ADC or a pharmaceutical composition comprising an ADC disclosed herein can result in a lower effective amount (e.g., concentration) of the drug than if the drug were 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.

[0088] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically 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 within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio. "Pharmaceutically acceptable carrier" may be a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in transporting or delivering the subject agent from one organ (or part of the body) to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissues of the patient (e.g., physiological compatibility, sterility, physiological pH, etc.). The term "carrier" denotes a natural or synthetic, organic or inorganic component that, when combined with the active ingredient, facilitates its application. The components of the pharmaceutical composition can also be co-mingled with the molecules of the present disclosure and with each other in such a way that no interactions that would substantially impair the desired pharmaceutical efficacy occur.Some examples of materials that can serve as pharmaceutically acceptable carriers are as follows: (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 carboxymethyl cellulose, methyl cellulose, ethyl cellulose, 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) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents 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) components of serum such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulations.

[0089] The pharmaceutical composition may be present, for convenience, in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. The term "unit dose" when used in relation to the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable as unitary dosages for the subject, each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.

[0090] The formulation of the pharmaceutical composition may depend on the route of administration. Injectable preparations suitable for parenteral administration, or for administration within the tumor, around the tumor, within the lesion or around the lesion, include, for example, sterile injectable aqueous or oily suspensions and 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 (such as solutions in 1,3 - propanediol or 1,3 - butanediol, etc.) in a parenterally acceptable non - toxic diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution (U.S.P.), and isotonic sodium chloride solution. In addition, sterile fixed oils have conventionally been employed as a solvent or suspending medium. For this purpose, any sterile fixed oil, including synthetic mono - or di - glycerides, may be employed. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations may be sterilized, for example, by filtration through a bacteria - retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other injectable sterile media prior to use.

[0091] Compositions suitable for oral administration may be presented as discrete units such as capsules, tablets, troches, etc., each containing a predetermined amount of the anti - inflammatory agent. Other compositions include suspensions in aqueous or non - aqueous liquids such as syrups, elixirs, or emulsions.

[0092] In some embodiments, the pharmaceutical composition used for therapeutic administration must be sterilized. Sterility is readily achieved by filtration through a sterilizing filter membrane (e.g., a 0.2 micron membrane). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The pharmaceutical composition will typically be stored in lyophilized form or as an aqueous solution if it is highly stable against heat denaturation and oxidative denaturation. The pH of the preparation will typically be from about 6 to 8, although higher or lower pH values may also be appropriate in certain cases.

[0093] "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 brain diseases described herein) required to produce a therapeutic effect in a subject (either alone or in combination with one or more other therapeutic agents). The effective amount will vary depending on factors recognized by those of ordinary skill in the art, including the specific disease being treated, the severity of the disease, the individual subject's parameters (including age, physical condition, size, sex, and weight), the duration of the treatment, the nature of any concomitant treatment (if any), the specific route of administration, and factors of the same kind within the knowledge and expertise of the medical practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with a small amount of routine experimentation. It is generally preferred that the maximum dose of an individual component or combination thereof, i.e., the highest safe dose in accordance with sound medical judgment, be used. However, it will be understood by those of ordinary skill in the art that a subject may, for medical, psychological, or any other factual reason, request a lower or tolerable dose.

[0094] Empirical considerations such as half-life will generally contribute to the determination of dosage. For example, therapeutic agents that are compatible with the human immune system, such as polypeptides that include regions from humanized or fully human antibodies, may be used to extend the half-life of the polypeptide and to prevent the polypeptide from being attacked by the host immune system. The frequency of administration may be determined and adjusted over the course of treatment and will generally be based on (but not necessarily limited to) the treatment, suppression, recovery, and / or retardation of the disease. Alternatively, sustained release formulations of the polypeptide may also be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0095] In some embodiments, dosing is daily, every other day, every 3 days, every 4 days, every 5 days, or every 6 days. In some embodiments, the dosing frequency is once per 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 per month, every 2 months, or every 3 months, or at longer intervals. The progress of this treatment can be readily monitored by conventional techniques and assays. The dosing regimen (including the anti-cancer agent used) can vary over time.

[0096] In some embodiments, for a normal weight adult subject, a dosage ranging from about 0.01 to 1000 mg / kg may be administered. In some embodiments, the dosage is between 1 and 200 mg. The specific dosing regimen, i.e., dosage, timing, and frequency (repetition), will depend on the specific subject and the subject's medical history, as well as the properties of the anti-cancer agent (such as the half-life of the anti-cancer agent and other considerations well known in the art).

[0097] For the purposes of the present disclosure, the appropriate dosage of the 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, prior treatments, the clinical history of the subject and response to the antagonist, as well as the discretion of the attending physician. Typically, the clinician will administer the anti-cancer agent until a dosage that achieves the desired result is reached. Administration of one or more anti-cancer agents may be continuous or intermittent and may depend, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the anti-cancer agent may be essentially continuous over a preselected period of time or, by way of example, may be a series of spaced doses either before, during, or after the onset of the disease.

[0098] 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, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, the symptom of the disease, or the predisposition to the disease.

[0099] The "subject" for which administration is contemplated refers to humans (i.e., males or females of any age group, by way of example, pediatric subjects (e.g., minors, children, or adolescent youths), or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)), or non-human animals. In some embodiments, the non-human animals are mammals (e.g., rodent animals (e.g., mice or rats), primate animals (e.g., cynomolgus monkeys or rhesus monkeys), commercially relevant mammals (e.g., cattle, pigs, horses, sheep, goats, cats, or dogs), or birds (e.g., commercially relevant birds such as chickens, ducks, geese, or pigeons)). The non-human animals can be male or female at any stage of development. The non-human animals can be transgenic animals or genetically modified animals.

[0100] In some embodiments, the subject is a companion animal (pet). "Companion animal" as used herein refers to pets and other domesticated animals. Non-limiting examples of companion animals include dogs and cats; domestic animals 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: rodent animals (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primate animals.

[0101] Reducing a disease (such as cancer) includes delaying the onset or progression of the disease or reducing the severity of the disease. Reducing a disease does not necessarily require a cured outcome. When used herein, "delaying" the onset of a disease means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be of variable length depending on the history of the disease and / or the individual being treated. A method of "delaying" or reducing the onset of a disease, or delaying the development of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a predetermined time frame and / or reduces the degree of the symptoms within a predetermined time frame as compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to give statistically significant results.

[0102] "Onset" or "progression" of a disease means the initial signs and / or subsequent progression of the disease. The onset of a disease is detectable and assessable using standard clinical techniques as is well known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of the symptoms. "Onset" includes occurrence, recurrence, and development. As used herein, "development" or "occurrence" of a disease includes initial development and / or recurrence.

[0103] Conventional methods known to those of ordinary skill in the art can be used to administer pharmaceutical compositions to a subject depending on the type of disease or the site of the disease to be treated. The pharmaceutical compositions can also be administered via other conventional routes, by way of example, orally, parenterally, by inhalation spray, topically, rectally, nasally, intraorally, intravaginally, or via an implantable reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, sub-sternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the pharmaceutical composition is administered via intravenous injection or infusion. Additionally, it can be administered to a subject via an injectable depot route of administration using, for example, 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 an intratumoral injection.

[0104] Example Introduction To date, pancreatic cancer (PC) remains one of the most lethal diseases, accounting for 56,770 deaths in the United States in 2019 and corresponding to 7% of all cancer mortalities. Despite the increasing research on immunotherapy and nano - medical treatments in recent years, the prognosis for PC patients is extremely poor, with less than 8% 5 - year survival. As an example, immune checkpoint blockade therapies, including cytotoxic T - lymphocyte - associated antigen 4 (CTLA4) inhibitors or programmed death ligand 1 (PD - L1) antibodies, have not shown sufficient clinical efficacy to treat PC patients to date. Innovative nano - medical formulations (such as liposomal docetaxel targeting EphA2) have also failed to provide clinical benefit in the treatment of advanced PC. These undesirable outcomes are largely attributed to the immunosuppressive tumor microenvironment (TME) of PC tumors, which is characterized by fibrotic stroma and insufficient angiogenesis. Such a TME creates a physical barrier that prevents T cells or nano - medicals from efficiently infiltrating the tumor and directly interacting with PC cells, leading to unfavorable effects. Developing new targeted therapeutics that can improve infiltrated PC tumors while maintaining strong tumor - specific efficacy highlights an unmet need.

[0105] Antibody-drug conjugates (ADCs) are a rapidly growing class of immunotherapeutics that have shown promising clinical efficacy against several cancers, including invasive solid tumors such as breast cancer that have an insufficient response to T cell immunotherapy. Unlike conventional chemotherapy, ADCs utilize chemical linkers that conjugate cytotoxic drugs to tumor-homing antibodies, and ADCs can selectively home to tumors while sparing normal tissues through recognition of tumor surface antigens and subsequent internalization and delivery of the cytotoxic drug into the targeted tumor cells. Compared to T cell immunotherapy (e.g., chimeric antigen receptor-T cell (CAR-T) or immune checkpoint blockade) or nanomedicine (e.g., liposomes or exosomes), ADCs are characterized by excellent tumor tissue penetration due to their ultra-small size (<10 nm), which is ~1,000 times smaller than the size of T cells and creates an excellent opportunity to increase drug delivery into stroma-dense PC tumors.

[0106] However, a major hurdle in the development of PC-targeted ADCs is to identify an immunotherapeutic target suitable for effectively distinguishing between PC and normal tissues. To meet the criteria for safety and efficacy for an optimal ADC, such a target needs to be at undetectable levels in normal tissues but abundantly presented on the cell surface of PC tumors, which allows it to be assayed against the tumor-homing antibody of the ADC. On the other hand, it is also required to facilitate rapid and robust cell internalization of the cytotoxic payload conjugated on the ADC. Several PC-targeted ADCs utilizing conventional PC targets (e.g., EGFR, EpHA2, and mesothelin) have been developed, but a systematic and quantitative comparison of established PC targets and other candidates at their cell surface protein levels has not yet been made. The present disclosure shows that performing such a fair and quantitative screening of cell surface proteins leads to the discovery of more optimal PC immunotherapeutic targets and promotes the development of PC-targeted ADCs.

[0107] ICAM1, also known as CD54, is a transmembrane glycoprotein of the immunoglobulin superfamily that is abnormally overexpressed in multiple types of cancer (e.g., triple-negative breast cancer) and is often associated with an invasive phenotype and a worse prognosis. In PC, ICAM1 is directly induced on pancreatic acinar cells by KRAS G12D mutations (the most common oncogenic mutations in 70 - 95% of PC patients), promotes the formation of neoplastic lesions in the pancreas, and leads to PC tumor initiation. This disclosure describes the identification and application of ICAM1 as a potential PC immunotherapy target based on a fair and quantitative screening algorithm. Therefore, an ICAM1 ADC was developed that induces potent and sustained PC tumor regression in vivo. To develop precision medicine, a non-invasive MRI approach was designed to identify ICAM1-expressing tumors suitable for ICAM1-targeted immunotherapy.

[0108] Results and Discussion ICAM1 is a rationally identified cell surface protein target for human PC. To identify suitable protein targets for distinguishing malignant PC tumors from normal tissues, a rationally designed cell surface protein target discovery algorithm was developed (Figure 1A). First, a fair and quantitative screening of 72 surface antigen panels related to cancer was performed in four established human PC cell lines (PANC-1, BxPC-3, Capan-1, and Capan-2), compared with two normal human pancreatic duct epithelial cells (HPDE and HPNE) as normal controls (Figure 1B). Among the 68 targets screened, 31 candidates were found to be commonly overexpressed in all four PC cell lines and were selected for further evaluation. By comparing their levels in human PC cells and normal pancreatic cells, ICAM1 emerged as the most overexpressed PC target among the top 10 candidates. In contrast, there was little expression in non-neoplastic HPNE and HPDE cells (Figure 1C). The cell surface density of ICAM1 was 3×10 on the four PC cell lines 5from 1×10 6 ranging up to 1×10 molecules / cells and is significantly higher than that of established PC targets (e.g., EGFR, MUC1, or EphA2). In addition, ICAM1 is ubiquitously overexpressed across all four human PC cell lines tested, suggesting that it would be a broad target population in PC patients. The overexpression of ICAM1 in human PC cells was further confirmed using immunofluorescence (IF) staining. ICAM1 is mainly expressed on the plasma membrane of four PC cell lines (PANC-1, BxPC-3, Capan-1, and Capan-2), but not in normal HNPE and HPNE cells (Figure 1D). This strong cell surface expression of ICAM1 on human PC cells makes it readily assessable for ICAM1-targeted immunotherapy (e.g., ADC or CAR-T cells).

[0109] To investigate whether ICAM1 high expression is a clinically relevant finding in human PC, immunohistochemistry (IHC) staining of ICAM1 was performed on 80 human PC tumor tissues and 20 normal pancreatic tissues. In Figures 1E and 1F, ICAM1 was constantly overexpressed on the plasma membrane and in the cytoplasm of PC cells from tumor tissues at various disease stages, while ICAM1 was completely absent in normal human pancreatic tissues. The degree of ICAM1 staining and the pathological score indicated that ICAM1 levels were positively correlated with the disease TNM stage (Figure 1G). For ICAM1-targeted immunotherapy, on-target but off-tumor sites in normal tissues were also evaluated. The protein level of ICAM1 was obtained from the Human Protein Atlas database ( proteinatlas.org) was investigated in an inclusive cohort of 45 normal human organs by inquiry. It was found that ICAM1 expression was absent in most normal tissues by IHC analysis, and only 4% (2 / 45, lung and kidney) of normal tissues showed high positive staining for ICAM1. This implies that the lung and kidney are potential sites that can be targeted but are not tumors for ICAM1-targeted immunotherapy. Moreover, previous studies have shown that ICAM1-targeted T cell immunotherapy induces neither acute nor delayed toxicity in both male and female athymic mice in an advanced thyroid cancer model.

[0110] The impact of ICAM1 overexpression on the clinical outcomes of PC patients was investigated by querying the R2: Genomics Analysis and Visualization Platform database ( hgserver1.amc.nl / , Datasheet: Mixed Pancreas Tumor-Zhang). The overall survival of PC patients with high ICAM1 expression was significantly worse than that of PC patients with low ICAM1 expression (Figure 1H, P = 0.021, log-rank test), which implies that ICAM1 may act as a clinical biomarker for poor prognosis in PC patients.

[0111] ICAM1 antibody recognizes and targets PC tumors in vivo. To evaluate ICAM1 as a potential immunotherapy target, the tumor specificity of an ICAM1 antibody in vivo was first determined in an orthotopic PC tumor model (Figure 2A). The ICAM1 monoclonal antibody was fluorescently labeled with the red fluorescent dye AF-647 and intravenously injected into PANC-1 tumor-bearing mice. AF-647-labeled IgG (IgG-AF) was used as an untargeted control. Since the in vivo fluorescent signal was interfered with by the location of the orthotopic PC tumors in the abdominal cavity and abdominal skin absorption, the animals were euthanized 24 hours after injection, and the PC tumors and the pancreatic tissues surrounding them were excised. Then, ex vivo imaging was performed to determine the tumor accumulation of the ICAM1-AF antibody. As observed in Figure 2B, the ICAM1 antibody selectively recognized and targeted the orthotopic PC tumors with high affinity compared to the untargeted IgG control. Normal pancreatic tissue adjacent to the PC tumors was not targeted by the ICAM1 antibody. Furthermore, its PC tumor specificity was confirmed. The quantified fluorescent signal (Figure 2C) confirmed that the tumor accumulation of the ICAM1 antibody after a single-dose tail vein administration was approximately 6-fold higher than that of the untargeted IgG-AF. These in vivo findings strongly support the development of an ICAM1 antibody-based immunotherapy with PC as the target.

[0112] Assuming that cell invasion activity is an essential factor in ADC design, the cellular internalization of the ICAM1 antibody in human PC cells was investigated using an imaging flow cytometry assay. As shown in Figure 2D, the phycoerythrin (PE)-conjugated ICAM1 antibody was robustly internalized by both PANC-1 cells and BxPC-3 cells via ICAM1 antigen-mediated endocytosis, while most of the PE-ICAM1 antibody was not internalized by normal HPNE cells due to significantly insufficient ICAM1 antigen expression. The internalization amount of the PE-ICAM1 antibody by human PC cells was quantified to be approximately 300-fold higher than that of HPNE cells (Figure 2E).

[0113] Next, the therapeutic causal relationship of blocking the ICAM1 signaling cascade in human PC cells was investigated using its neutralizing antibody. In Figure 2F, treatment with the ICAM1 neutralizing antibody (2 μg / mL) did not clearly alter the proliferation of either PANC-1 cells or BxPC-3 cells. However, the ICAM1 neutralizing antibody strongly inhibited the migration of PANC-1 cells and BxPC-3 cells and reduced the cell migration of PANC-1 cells and BxPC-3 cells by 39% and 44%, respectively, compared to the IgG control (Figure 2G). Correlatively, the ICAM1 neutralizing antibody has also been reported to strongly inhibit PC carcinogenesis initiation in vivo. These findings indicate that ICAM1 can function as a PC tumor homing target. They also indicate that targeting the ICAM1 signaling cascade can also prevent disease progression.

[0114] Rational design of ICAM1 antibody-drug conjugates To change the ICAM1 target to PC treatment, a rationally designed ICAM1 ADC was designed as an immunotherapy for PC-targeted treatment (Figure 3A). Considering that the chemical linker and cytotoxic payload substantially affect the efficacy of the ADC, the first step was to select the optimal ADC formulation for PC treatment using a fair and quantitative screening approach. A series of ICAM1 ADCs were modified with four clinically tested ADC linkers and cytotoxic payloads (SMCC-DM1, Vc-MMAE, Mc-MMAF, duocarmycin) at an equal drug-to-antibody ratio (DAR), and their cytotoxicity against human PC cells was compared in comparison with an unlabeled IgG ADC control. As shown in Figure 3B, ICAM1-SMCC-DM1 showed the lowest IC50 (38.1 nM) among the four ADC formulations tested in the treatment of PANC-1 cells (the others had IC50: 83.9 - 240.4 nM). The IC50 of ICAM1-SMCC-DM1 was more than 2,000 times lower than that of Gem (89.1 μM), the first-choice chemotherapeutic agent for PDAC treatment. SMCC-DM1 is a clinically effective ADC formulation consisting of a non-cleavable chemical linker and mertansine (DM1), a potent microtubule inhibitor. Therefore, SMCC-DM1 was selected as the optimized ADC formulation, and subsequently, ICAM1-SMCC-DM1 (ICAM1-DM1) was synthesized as an ICAM1 ADC optimized for PC-targeted treatment. IgG-SMCC-DM1 (IgG-DM1) was also prepared under the same experimental conditions as the unlabeled control. The drug-to-antibody ratios (DARs) for ICAM1-DM1 and IgG-DM1 were controlled by the input amounts of DM1 and the antibody and achieved 3.4 for ICAM1-DM1 and 3.2 for IgG-DM1 as determined using a UV / VIS spectroscopic assay.

[0115] ICAM1-DM1 selectively eliminates PC cells in vitro and in vivo. The PC-specific cytotoxicity of ICAM1-DM1 was determined in two human PC cells (PANC-1 and BxPC-3) and normal HPNE cells (Figure 3C-3E). The first-choice chemodrug GEM and non-targeted IgG-DM1 were used as controls. As observed in Figure 3D and Figure 3E, ICAM1-DM1 showed potent cytotoxicity against PANC-1 cells and BxPC-3 cells. The IC50 of ICAM1-DM1 was determined to be 9.8 nM for PANC-1 and 4.0 nM for BxPC-3, which was significantly lower than those of GEM and IgG-DM1 (30 nM-88 μm). Moreover, ICAM1-DM1 did not show cytotoxicity in normal HPNE cells due to their insufficient ICAM1 expression (Figure 3E). These in vitro results strongly support the evaluation of the antitumor activity of ICAM1-DM1 in the in vivo setting of a PC model.

[0116] The antitumor activity of ICAM1-DM1 was examined in the in vivo suppression of orthotopic PC tumor growth (Figure 3F). ICAM1-DM1 or IgG-DM1 (non-targeted control) was administered intravenously at 15 mg / kg every 3 weeks to PANC-1-Luc tumor-bearing mice. In comparison, GEM was administered intravenously at a dose of 5 mg / kg weekly due to its short circulatory half-life (0.28 hr). After two ADC injections, the ICAM1-DM1 treatment group exhibited potent and sustained tumor regression compared to the other groups (Figures 3G - 3H). Quantified tumor volume showed that ICAM1-DM1 significantly reduced PC tumor growth by up to 49% compared to the PBS (mock) group (Figure 3I). The mechanism by which ICAM1-DM1 induces toxicity was further investigated by measuring the expression of the cell proliferation marker Ki67 in PC tumor tissue. As observed in Figures 3K and 3L, the Ki67-positive cell population in the ICAM1-DM1 treatment group, which contributed to potent and sustained tumor suppression, was significantly reduced compared to the other groups. This potent antitumor activity of ICAM1-DM1 also effectively inhibited spontaneous PC metastasis to normal organs (including the lung, liver, and spleen) (Figures 3M and 5). There was no evidence of histopathological damage to normal vital organs collected from the ICAM1-DM1 treatment group.

[0117] Non-invasively evaluate the ICAM1 expression of tumors by MRI. An MRI-based molecular imaging approach for precision medicine was developed to non-invasively and rapidly identify PC patients who would benefit from ICAM1-targeted immunotherapy. In clinical practice, needle biopsy is commonly introduced to examine the adequacy of target expression in tumor tissue prior to targeted therapy, but this approach is limited by the complexity and heterogeneity within tumors and its invasiveness and low accuracy (<50%). To overcome these obstacles, an ICAM1-targeted MRI probe was developed and used to map tumor ICAM1 expression in an orthotopic PC model using MRI (Figure 4A). The ICAM1-targeted MRI probe was first modified by covalently conjugating an ICAM1 antibody to DTPA-Gd (a clinically used MRI contrast agent). IgG-Gd was prepared as a non-targeted control. Then, ICAM1-Gd or IgG-Gd was administered intravenously at a dosage of 5 mg / kg mouse weight into ICAM1-expressing PANC-1 tumor-bearing mice. In vivo MRI was performed on PC tumor-bearing mice with a series of MRI sequences including T1- and T2-weighted spin echo imaging before injection of the MRI probe and 24 hours after its injection. In Figure 4B, PC tumors (yellow circles) in the peritoneal cavity were located by analyzing high-resolution T2-weighted MRI images. Once the PC tumors were located, T1-weighted MRI images were used to quantitatively measure the MRI signal change in the area of the PC tumor (yellow circle) as a function of gadolinium accumulation within the tumor from the administered MRI probe. In Figure 4C, the T1 MRI signal of the tumor increased by ~50% in the ICAM1-Gd group, while there was no MRI signal change in the non-targeted IgG-Gd group (n = 3 / group). These MRI signal changes are positively correlated with the level of antigen expression on the targeted tumor, which can be used to identify ICAM1-positive patients who would benefit from ICAM1-targeted immunotherapy.

[0118] In summary, the present disclosure provides experimental evidence that ICAM1 is a suitable ADC target for human PC. The utility of this target can also be extended to the development of other immunotherapeutic agents, including CAR T cells or bispecific antibodies directed to ICAM1.

[0119] Materials and Methods Materials. The purified anti-human CD54 antibody (Clone: HCD54), phycoerythrin (PE)-conjugated mouse anti-human ICAM-1 antibody (PE-ICAM1), and PE-conjugated mouse IgG isotype (PE-IgG) were purchased from BioLegend (San Diego, CA, USA). G-DM1, G-MMAE, G-MMAF, G-Duoca for pre-screening ADC were purchased from Levena Biopharma (San Diego, CA). SMCC-DM1 was purchased from Medkoo (Morrisville, NC, USA). Zeba (trademark) Spin Desalting Columns, (7K MWCO), Alexa Fluor 647 NHS ester, Lab-Tek II Chamber Slide System, ProLong Gold Antifade Mountant were obtained from Thermo Fisher Scientific. Gemcitabine hydrochloride (GEM), gadolinium(III) chloride hexahydrate (GdCl3·6H2O), diethylenetriaminepentaacetic acid dianhydride (DTPAA), sodium bicarbonate, trisodium citrate dihydrate were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco PBS, DAPI, Quant-iT RNA Assay Kit, 0.25% trypsin / 2.6 mM EDTA solution, Gibco DMEM, Gibco DMEM / F12 (1:1), Roswell Park Memorial Institute (RPMI)-1640 medium, and McCoy's 5A medium were purchased from Invitrogen (Carlsbad, CA). MEGM Mammary Epithelial Cell Growth Medium was purchased from Lonza (Basel, Switzerland). Quantum Simply Cellular microbeads were purchased from Bangs Laboratory (Fishers, IN). Dojindo Cell Counting Kit CCK-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA).The human pancreatic cancer tissue and normal tissue array (PA1002a) was purchased from US Biomax (Derwood, MD).

[0120] Cell culture. PANC-1 cells, BxPC-3 cells, Capan-1 cells, Capan-2 cells, and HPNE cells were purchased from ATCC (Manassas, VA). HPDE cells were purchased from Kerafast (Boston, MA). PANC-1, Dulbecco's modified Eagle's medium with 10% FBS; BxPC-3, RPMI-1640 medium with 10% FBS; Capan-1, Iscove's modified Dulbecco's medium with 20% FBS; Capan-2, McCoy's modified 5a medium with 10% FBS; HPNE, 75% DMEM (without glucose, with additional 2 mM L-glutamine and 1.5 g / L sodium bicarbonate), 25% Medium M3 Base (5% FBS, 10 ng / ml human recombinant EGF, 5.5 mM D-glucose (1 g / L), 750 ng / ml puromycin); HPDE, Keratinocyte Basal Medium + supplied supplements (Lonza, Clonetics KBM, Cat# CC-3111). All cells were maintained at 37 °C in a humidified incubator with 5% (vol / vol) CO2.

[0121] Quantification of ICAM-1 surface expression. Pancreatic cancer cell ICAM1 surface protein expression was evaluated by a BD FACSCalibur flow cytometer (BD Biosciences) as previously described. Quantification of the density of ICAM-1 on the cell surface was determined by reference to Quantum Simply Cellular microbeads using the protocol provided by the manufacturer. 10 6Cells were collected and washed twice by passing through a suspension dehydration cycle. The cells were blocked with 1% BSA in PBS for 30 min in an ice bath. After blocking with BSA, the cells were incubated with phycoerythrin (PE)-conjugated ICAM1 antibody for 1 h at room temperature. The cells were washed three times with 1% BSA in PBS and resuspended in PBS for evaluation by flow cytometry.

[0122] Immunohistological staining. Immunohistochemical studies were performed on paraffin-embedded human PDAC and normal tissue microarrays (PA1002a, US Biomax). Microarray samples of 40 cases of human PDAC tissue and 10 cases of human normal tissue were evaluated for ICAM-1 expression as previously described. Individual tissue scores in the microarray were scored by a surgical pathologist blinded to the identity of the samples. Immunostaining was scored by calculating the H score. In the H score, the percentages of strongly (3+), moderately (2+), and weakly (1+) cell staining were multiplied according to the formula: H score = 3 × (% cell staining 3+) + 2 × (% cell staining 2+) + 1 × (% cell staining 1+). Micrographs were taken on an Olympus BX41 microscope by using an Olympus Q-Color5 digital camera (Olympus America Inc.).

[0123] In vitro binding and internalization of ICAM-1 Ab The specific binding of ICAM1 antibody to human pancreatic cancer cell lines in vitro was assayed using a PE-ICAM1 antibody. IgG was used as a control. Cells were plated at 5×10 in an 8-well chamber slide 3Seeded at the density of cells / well. After allowing to recover for 24 hours, the whole medium was replaced with that containing 1% FBS with PE-ICAM1 antibody. The cells were further incubated in the PE-ICAM1-containing medium at 37 °C for 4 hours. Subsequently, the cell monolayer was rinsed with cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS solution. The 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).

[0124] In the imaging flow cytometry study, cells were seeded in 6-well chamber slides at a density of 1×10 6 Seeded at the density of cells / well. After 24-hour cell recovery, the whole medium was replaced with that containing 1% FBS with PE-ICAM1 antibody. The cells were incubated in the PE-ICAM1-containing medium at 37 °C for 1 hour. Subsequently, the cell monolayer was collected, rinsed twice with cold PBS and resuspended, and evaluated using an Amnis imagestreamX Mark II imaging flow cytometry (Luminex, Austin, TX, USA).

[0125] Therapeutic effect of ICAM-1 Ab The in vitro therapeutic effect of ICAM1 antibody on human pancreatic cancer cell lines was assayed using quantitative phase contrast imaging. IgG was used as a control. Cells were seeded in 6-well plates at 5×10 4Seeded at the density of cells / well. After allowing to recover for 24 hours, the whole medium was replaced with that containing ICAM1 antibody or IgG at a dosage of 2 μg / mL. The cells were incubated at 37 °C for 24 hours in the medium containing ICAM1 or IgG. Thereafter, the plate was placed under a quantitative phase contrast imaging microscope (Holomonitor M4, Phase Holographic Imaging Phi AB, Lund, Sweden) provided in the incubator, and further imaged at 5-minute intervals for 24 hours. Subsequently, cell motility, morphology, and proliferation were analyzed using Hstutio4.

[0126] Preparation and characterization of ADC Ab-Gd. IgG-DM1 and ICAM1-DM1 were prepared by mixing IgG or ICAM1 Ab (2.4 mg, 16 nmol) with SMCC-DM1 (1.2 mg, 1.1 μmol) in phosphate buffer (pH 7.2) at room temperature with rotation for 1 h. Free SMCC-DM1 was removed by Ultra-4 Centrifugal Filter (30K MWCO). IgG-DM1 and ICAM1-DM1 were washed several times with PBS (pH 7.4) and redispersed in PBS.

[0127] The ADC was characterized by UV / Vis spectroscopy, and the antibody-drug ratio was calculated according to the equation: ADR = C 薬物 / C Ab =(A 280 ε λ(D)mAb -A λ(D) ε 280mAb )(A 280 ε λ(D)薬物 -A λ(D) ε 280薬物 ) / [(ε 280薬物 ε λ(D)mAb -ε λ(D)薬物 ε 280mAb )(ε 280mAb ε λ(D)薬物 -ε λ(D) m Ab ε 280薬物 )] Calculated according to.

[0128] IgG-DTPA-Gd and ICAM1-DTPA-Gd were prepared as previously reported. DTPAA (0.4 mg, 1.1 μmol) was slowly added to IgG or ICAM1 Ab (0.2 mg, 1.3 nmol) in NaHCO3 buffer (pH 9.0, 0.1 M), and the mixture was rotated overnight at room temperature. DTPA-conjugated IgG or ICAM1 Ab was purified by Ultra-4 Centrifugal Filter (30K MWCO) and redispersed in citrate buffer (pH 6.5, 0.1 M). Then GdCl3 (0.1 mg, 0.27 μmol) in 0.1 M citrate buffer (pH 6.5) was mixed with DTPA-conjugated IgG or ICAM1 Ab by rotating at room temperature for 24 h. Free Gd 3+ was removed by Ultra-4 Centrifugal Filter (30K MWCO), and IgG- or ICAM1-DTPA-Gd was redispersed in PBS for subsequent use. Ab-Gd was characterized by liquid chromatography electrospray ionization mass spectrometry LC-MS.

[0129] Cytotoxicity assay. Human pancreatic cancer cell lines were seeded in 96-well plates at a density of 5×10 3 cells / well and allowed to adhere overnight. Then the culture medium was replaced with medium containing free GEM, IgG, or ICAM1-conjugated DM1, Duo, MMAE, MMAF at various drug concentrations. After culturing the cells for another 72 h, cytotoxicity was determined by CCK-8 assay following the protocol provided by the supplier. After removing the drug-containing medium, the cells were carefully washed with PBS, followed by the addition of CCK-8-containing medium solution. Then the cells were incubated in CCK-8 medium for 4 h. The plate was read at an absorbance wavelength of 450 nm using a microplate reader (Synergy2; BioTek, Winooski, VT, USA). Cell viability was determined by comparing the absorbance of cells incubated with the drug to the absorbance of control cells incubated without the drug.

[0130] Orthotopic PDAC mouse model. All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children's Hospital. PANC-1 cells were transfected with a plasmid expressing luciferase and GFP genes according to the manufacturer's instructions (MGH, Boston). The success of transfection was confirmed 72 hours after infection by visualization of GFP under a fluorescence microscope. Stably transfected cells were sorted by flow cytometry for GFP signal twice using FACSAria II flow cytometry (BD Biosciences) and maintained in DMEM-10% FBS. Orthotopic pancreatic cancer models were prepared by injecting PANC-1 cells into the pancreas of 8-week-old male thymus-deficient nude mice (Charles River; n = 6 per group) using an established surgical procedure. Mice were anesthetized with isoflurane (5% with O2) during surgery. An incision was made in the left flank abdominal region where the pancreas is typically located posterior to the center of the spleen. The pancreas was then gently pulled out using forceps, and 50 μL of 1×10 6PANC-1 cells were carefully injected into the pancreas. After injection, the pancreas was placed in the back of the abdominal cavity and in front of the abdominal muscles, and the skin was closed with 4-0 Polysorb sutures and surgical staples. The treatment was started after a 2-week recovery. The animals were randomly divided into four groups (n = 6): PBS control, treatment with gemcitabine (GEM), treatment with non-targeted IgG-DM1, and treatment with ICAM1-DM1. Mice were treated via intravenous tail vein injection at a dose of 12 mg / kg mouse weight per 3 weeks for IgG-DM1 and ICAM1-DM1, and at a dose of 80 mg / kg mouse weight twice a week for GEM, while the control group received only PBS injection. In total, there were two injections at 3-week intervals for the ADC treatment group and the control group, and 12 injections at 3- or 4-day intervals for GEM. Body weight was measured twice a week, and tumor growth after the mice received an i.p. injection of D-luciferin was monitored using an IVIS Spectrum Imaging System (PerkinElmer).

[0131] MRI In Vivo. In vivo MRI was performed on tumor-bearing mice in two groups that were intravenously injected with IgG-Gd and ICAM-Gd (at a dosage of 5 mg / kg mouse weight), respectively. Images were obtained on a 9.4 T Bruker Horizontal Bore MRI with a fast spin echo sequence for T1- and T2-weighted MRI before injection and 24 hours after injection. The imaging parameters were as follows: for T2-weighted imaging, a repetition time (TR) of 1,523 ms, an echo time (TE) of 33 ms, a matrix of 340×220, a field of view of 40×28-mm2, a flip angle of 180°, and a slice thickness of 0.6 mm; for T1-weighted imaging, a TR of 700 ms and a TE of 22 ms. To quantify the tumor signal intensity, a region of interest (ROI) was drawn to surround the entire tumor in the same slice at the same imaging depth. Pixel intensity was calculated by ImageJ software and normalized to the area of the ROI.

[0132] Histology. Organs (liver, spleen, kidney, pancreas, heart, lung, and muscle), as well as tumor samples, were collected at the endpoint. The pathology of orthotopic PANC-1 tumors treated with ICAM-DM1, IgG-DM1, GEM, and PBS was investigated by H&E staining, Ki67 staining, and ICAM1 immunohistological staining. All stainings were performed on tumor sections following standard protocols.

[0133] Statistical analysis. Quantitative data are presented as mean ± SD. Differences were compared using an unpaired t-test. Statistics were performed using Microsoft Excel software. A P-value ≤ 0.05 was considered statistically significant.

[0134] Examples of linker and drug structures in ADC The structures of the linkers and drugs used in this disclosure are provided below.

[0135] Name: SMCC-DM1 Chemical name: N2'-Deacetyl-N2'-[3-[[1-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]-2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]-maytansine

[0136] Chemical structure:

Chem.

[0137] Name: VC-MMAE (MC-VC-PAB-MMAE) Chemical Name: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0138] Chemical Structure:

Chem.

[0139] Name: MC-MMAF Chemical Name: ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0140] Chemical Structure:

Chem.

[0141] Name: Mal-PEG4-VC-PAB-DMEA-Seco-duocarmycin Chemical Name: Methyl (8S)-4-[2-[[4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methoxycarbonyl-methylamino]ethyl-methylcarbamoyl]oxy-8-(chloromethyl)-6-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-7,8-dihydro-3H-pyrrolo[3,2-e]indole-2-carboxylate

[0142] Chemical Structure:

Chem.

[0143] Equivalents 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.

[0144] Articles such as "a", "an", and "the" may mean one or more than one, unless indicated to the contrary or otherwise apparent from the context. A claim or recitation that includes "or (or / alternatively)" among two or more members of a group is considered to be satisfied if one, more than one, or all of the members of the group are present, unless indicated to the contrary or otherwise apparent from the context. The present disclosure of a group that includes "or (or / alternatively)" among two or more members of the group provides for the situation where there is exactly one member of the group, the situation where there are more than one members of the group, and the situation where all members of the group are present. For the sake of brevity, these situations are not described in detail herein individually, but it will be understood that each of these situations is provided herein and may be specifically claimed or disclaimed.

[0145] 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 related recitations are incorporated into another claim. For example, a claim (which is dependent on another claim) may be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Further still, when a claim recites a composition, a method of making or using the composition that follows any of the methods of making or using disclosed herein or, if any, methods known in the art will also be understood to be included, unless otherwise indicated or it would be apparent to one of ordinary skill in the art that a conflict or inconsistency would result.

[0146] When elements are presented as a listing (e.g., in the form of a Markush group), it should be understood that any sub - groups of the possible elements are also disclosed, and which elements or sub - groups of elements can be removed from the group. It should also be noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. Generally, when an aspect, product, or method is referred to as including specific elements, features, or steps, it should be understood that aspects, products, or methods consisting of or consisting essentially of such elements, features, or steps are also provided. For the sake of brevity, those aspects are not individually described in detail herein, but it will be understood that each of these aspects is provided herein and may be specifically claimed or disclaimed.

[0147] When a range is given, the endpoints are included. Further, unless otherwise indicated or otherwise apparent from the context and / or the understanding of one of ordinary skill in the art, values expressed as a range are to be considered to include any specific value within the stated range, in some aspects, down to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For the sake of brevity, the values within each range are not individually described in detail 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 apparent from the context and / or the understanding of one of ordinary skill in the art, it should also be understood that values expressed as a range may also include any sub - range within the given range (where the endpoints of the sub - range are expressed with the same degree of precision as one tenth of the unit of the lower limit of the range).

[0148] When a website is provided, the URL address is provided as browser - non - executable code, and the periods of each web address are shown in parentheses. The actual web address does not contain parentheses.

[0149] In addition, it should be understood that any specific aspect of the present disclosure may be explicitly excluded from any one or more of the claims. When a range is given, any value within the range may also be explicitly excluded from any one or more of the claims. Any aspect, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more of the claims. For the sake of brevity, all aspects in which one or more elements, features, objects, or aspects are excluded are not explicitly represented herein.

Claims

**Claim 1** A method of treating pancreatic cancer, the method 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. **Claim 2** The method of claim 1, wherein the drug is selected from the group consisting of N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin. **Claim 3** The method of claim 2, wherein the drug is DM1. **Claim 4** The method according to any one of claims 1 to 3, wherein the ICAM1 antibody and the drug are conjugated via a linker. **Claim 5** The method of claim 4, wherein the linker is a cleavable linker. **Claim 6** The method of claim 5, 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), acetyl butyrate, and CL2A. **Claim 7** The method of claim 6, wherein the cleavable linker is Val-cit. **Claim 8** The method of claim 4, wherein the linker is a non-cleavable linker. **Claim 9** The method 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 maleimidomethyl cyclohexane-1-carboxylate (MCC). **Claim 10** The method of claim 8, wherein the non-cleavable linker is an N-succinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker. **Claim 11** The method according to any one of claims 1 to 10, wherein the ICAM1 antibody is selected from the group consisting of IgG, Ig monomer, Fab fragment, F(ab')2 fragment, Fd fragment, scFv, scAb, dAb, Fv, affibody, bispecific antibody, single domain heavy chain antibody, and single domain light chain antibody. **Claim 12** The method according to any one of claims 1 to 11, wherein the ICAM1 antibody is enlimomab or HCD54.

13. The method according to any one of claims 1 to 12, wherein the ratio of the ICAM1 antibody to the drug in the ADC is from 1:1 to 1:

10.

14. The method according to claim 13, wherein the ratio of the ICAM1 antibody to the drug in the ADC is 1:

4.

15. The method according to any one of claims 1 to 14, wherein the ADC is administered via injection.

16. The method according to claim 15, wherein the injection is intravenous injection or intratumoral injection.

17. A method for treating pancreatic cancer, the method 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 N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1) via an N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

18. A method for predicting the responsiveness of a subject with pancreatic cancer to treatment with an antibody-drug conjugate (ADC) comprising an ICAM1 antibody or an intercellular adhesion molecule 1 (ICAM1) antibody conjugated to a drug, the method comprising: (i) administering to the subject an effective amount of an ICAM1 antibody labeled with an imaging agent; (ii) visualizing the tumor via imaging; and (iii) determining the level of ICAM1 on the tumor comprising, wherein a higher level of ICAM1 indicates that the subject will respond to treatment with the ICAM1 antibody or ADC as compared to a subject having a lower level of ICAM1.

19. The method according to claim 18, wherein the ICAM1 antibody in (i) is labeled with DTPA-Gd.

20. The method according to claim 18 or claim 19, wherein the visualization in (ii) is via magnetic resonance imaging (MRI).

21. The method according to any one of claims 18 to 20, further comprising administering an effective amount of an ICAM1 antibody or ADC to a subject predicted to respond to treatment for treating pancreatic cancer.

22. The method according to any one of claims 18 to 21, wherein the drug is selected from the group consisting of: N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) mertansine (DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and duocarmycin.

23. The method according to claim 22, wherein the drug is DM1.

24. The method according to any one of claims 18 to 23, wherein the ICAM1 antibody and the drug are conjugated via a linker.

25. The method according to claim 24, wherein the linker is a cleavable linker.

26. The method according to claim 25, 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), acetyl butyrate, and CL2A.

27. The method according to claim 24, wherein the cleavable linker is Val-cit.

28. The method according to claim 24, wherein the linker is a non-cleavable linker.

29. The method according to claim 28, wherein the non-cleavable linker is selected from the group consisting of: N-succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC), and maleimidomethyl cyclohexane-1-carboxylate (MCC).

30. The method according to claim 29, wherein the non-cleavable linker is an N-succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker.

31. The method according to any one of claims 18 to 30, wherein the ICAM1 antibody is selected from the group consisting of: IgG, Ig monomer, Fab fragment, F(ab')2 fragment, Fd fragment, scFv, scAb, dAb, Fv, affibody, bispecific antibody, single domain heavy chain antibody, and single domain light chain antibody.

32. The method according to any one of claims 18 to 30, wherein the ICAM1 antibody is enlimomab or HCD54.

33. The method according to any one of claims 18 to 32, wherein the ratio of the ICAM1 antibody to the drug in the ADC is from 1:1 to 1:

10.

34. The method according to claim 33, wherein the ratio of the ICAM1 antibody to the drug in the ADC is 1:4.

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