Fc-engineered monoclonal antibodies resistant to tumor immunosuppressive ICAM-1 / CD54

Abstract

The ICAM-1 / CD54 protein has been shown to be a suppressor of antibody-mediated humoral immunity, promoted by direct binding to the CH3 domain within the IgG1 heavy chain. This binding inhibits the immune effector activity of antibodies by reducing IgG1 binding to Fc receptors and the C1q complement initiator protein on effector cells. Furthermore, this binding may reduce the activity of antibody-drug conjugates by delaying target cell internalization. Engineered antibodies with modified CH3 regions can be created that reduce or eliminate ICAM-1 / CD54 binding, thereby resulting in antibodies with improved immune effector activity and improved killing of antibody-drug conjugates in the presence of ICAM-1 / CD54. These antibodies are useful for treating patients with cancer, inflammatory diseases, and infectious diseases in which ICAM-1 / CD54 levels are elevated. TIFF2025528832000005.tif68170

Description

[Technical Field]

[0001] Technical field of the invention The present invention relates to the fields of humoral immunity and humoral immuno-oncology. In particular, the present invention relates to methods and compositions of agents for overcoming the immunosuppressive effects of ICAM-1 / CD54 proteins and improving the efficacy of antibody-based therapies that inhibit cancer cell proliferation and other humoral immunosuppressive diseases.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (008966.00037 sequence listing.xml, size: 15 KB, created on July 31, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Background of the Invention Humoral immunity is the main mechanism by which vertebrate host organisms monitor and defend against dysregulated and transformed host cells. In cancer biology, immune checkpoint inhibitors that can overcome suppressed cellular immunity show strong effects in eliciting the killing effect of activated CD8+ T cells against tumor subsets (Hodi FS, et al. N Engl J Med 363:711-723, 2010 (Non-Patent Document 1)). Several commercially approved therapeutic antibodies have been reported to exert tumor-killing effects via humoral-mediated antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015 (Non-Patent Document 2); Ruck T, et al. Int J Mol Sci. 16:16414-16439, 2015 (Non-Patent Document 3); Pelaia C, et al. Biomed Res Int 4839230:1-9, 2018 (Non-Patent Document 4)). Recent parallel findings have shown that tumors produce factors that suppress humoral immune pathways and thus inhibit ADCC-, ADCP-, and CDC-mediated tumor killing (Vergote I, et al. J Clin Oncol 34:2271-2278 (Non-Patent Document 5); Kline JB, et al. J Clin Oncol 5:15, 2018 (Non-Patent Document 6); Wang W et al. Cytogenet Genome Res 152:169-179, 2017 (Non-Patent Document 7); Kline JB et al. Eur J Immunol. 48:1872-1882, 2018 (Non-Patent Document 8); Grasso L et al. Oncol Letters 23:2, 2022 (Non-Patent Document 9)). Furthermore, these tumor-produced factors have been shown to bind to the antibody component within antibody-drug conjugates (ADCs), reducing their internalization and overall target cell killing (Nicolaides NC, et al. PloS ONE DOI.org / 10.1371 / journal.pone.0285161, 2023).

[0004] Antibody-mediated humoral immune responses are controlled by the regulated binding of antibodies to cell surface antigens, which places the antibody on the antigen epitope in constant proximity to the cell surface. Once bound, antibodies bind to the Fc-γ activating receptors FCGR3A (CD16a) and FCGR2A (CD32a) on natural killer (NK) cells or dendritic / myeloid / monocyte cells, respectively, to initiate ADCC or antibody-dependent cellular phagocytosis (ADCP) (cells involved in ADCC are referred to herein as "immune effector cells") and potentially bind to the C1q complement initiation protein to cause target cell death of antibody-bound cells via the classical complement CDC pathway (Reuschenbach M, et al. Cancer Immunol Immunother 58:1535-1544, 2009). These effects have been observed with several therapeutic antibodies, including but not limited to rituximab, trastuzumab, cetuximab, pertuzumab, daratumumab, and alemtuzumab (Zhou X, et al. Oncologist 13:954-966, 2008 (Non-Patent Document 12); Hsu YF, et al. Mol Cancer 9:1-8, 2010 (Non-Patent Document 13); Spiridon CI, et al. Clin Cancer Res 8:1720-1730, 2002 (Non-Patent Document 14); Luo C, et al. Sci Rep 7:46347, 2017 (Non-Patent Document 15); Casneuf T et al. Blood Adv 1:2105-2114, 2017 (Non-Patent Document 16)).

[0005] Multiple reports have revealed that tumor-produced soluble and membrane-bound forms of ICAM-1 are associated with poor prognosis in patients with gastric cancer, NSCLC, melanoma, breast cancer, colorectal cancer, multiple myeloma, and lymphoma (Maruo Y et al. Int J Cancer 100:486-490, 2002; Wu M et al. Path Res Pract 10.1016 / j.prp.2020.153029; Roland CL et al. Surgery 141:705-707, 2007). Some of these cancer indications are treated with antibody-based therapies that exploit immune effector mechanisms for tumor killing.

[0006] There is a continuing need to develop means and agents that may overcome humoral immunosuppression and / or reduced efficacy of ADCs mediated by soluble and membrane-bound forms of ICAM-1 / CD54 (referred to herein as ICAM-1) in cancer patients and other diseases in which ICAM-1-associated humoral immunosuppression occurs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hodi FS, et al. N Engl J Med 363:711-723, 2010 [Non-patent document 2] DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015 [Non-patent document 3] Ruck T, et al. Int J Mol Sci. 16:16414-16439,2015 [Non-patent document 4] Pelaia C, et al. Biomed Res Int 4839230:1-9,2018 [Non-Patent Document 5] Vergote I, et al. J Clin Oncol 34:2271-2278

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[0008] One aspect of the present invention is an immunosuppression-resistant human IgG1 antibody that has 1 to 4 amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody, whereas an immunosuppression-sensitive human IgG antibody does not have these 1 to 4 amino acid substitutions. The immunosuppression of immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0009] Another aspect of the present invention is a polynucleotide encoding an immunosuppression-resistant human IgG1 antibody. The immunosuppression-resistant human IgG1 antibody has one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody, while the immunosuppression-sensitive human IgG1 antibody does not have these one to four amino acid substitutions. The immunosuppression of the immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0010] Another aspect of the present invention is a nucleic acid vector encoding an immunosuppression-resistant human IgG1 antibody. The immunosuppression-resistant human IgG1 antibody has one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody, while the immunosuppression-sensitive human IgG1 antibody does not have these one to four amino acid substitutions. The immunosuppression of the immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0011] Another aspect of the present invention is a stable cell line comprising a nucleic acid vector. The stable cell line expresses an immunosuppression-resistant human IgG1 antibody. The immunosuppression-resistant human IgG1 antibody has one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody, while the immunosuppression-sensitive human IgG1 antibody does not have these one to four amino acid substitutions. The immunosuppression of the immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0012] Yet another aspect of the present invention is a method for treating a disease in a patient in which the expression level of ICAM-1 / CD54 is elevated compared to that of a healthy human population. An immunosuppression-resistant human IgG1 antibody is administered to the patient in a canonical or antibody-drug conjugate (ADC) format. The immunosuppression-resistant human IgG1 antibody has one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody, while the immunosuppression-sensitive human IgG1 antibody does not have these one to four amino acid substitutions. The immunosuppression of the immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0013] Another aspect of the present invention is a method for treating a patient with cancer or an inflammatory disease. A full-length human IgG1 antibody is administered to the patient with cancer or an inflammatory disease. The antibody comprises a heavy chain with one to four amino acid substitutions in Kabat regions 367-425, preferably in Kabat regions 369-410, located in the heavy chain of the full-length human IgG1 antibody. In one embodiment, the amino acid substitutions occur at amino acid residues YSKL (407-410) (SEQ ID NO: 3).

[0014] Yet another aspect of the present invention is a screening method for identifying candidate antibodies resistant to ICAM-1 / CD54 immunosuppression. (a) A human cancer cell line expressing an antigen is contacted with a candidate IgG1-type antibody that specifically binds to the antigen. The contacting is performed in the presence of ICAM-1 / CD54 (SEQ ID NO: 1). (b) A human cancer cell line expressing an antigen is contacted with a candidate IgG1-type antibody that specifically binds to the antigen. The contacting is performed in the absence of ICAM-1 / CD54 (SEQ ID NO: 1). (c) The antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) of the human cancer cell line stimulated by the candidate antibodies in steps (a) and (b) is determined. (d) The antibody may be prepared in an antibody-drug conjugate (ADC) format. Target cell killing is determined by comparing the amount achieved in step (a) with the amount achieved in step (b).

[0015] Another aspect of the present invention is a method for identifying candidates resistant to ICAM-1 / CD54 immunosuppression by screening candidate immunosuppression-resistant human IgG1 antibodies having one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody. The candidate immunosuppression-resistant human IgG1 antibody is contacted with biotinylated human CD16a Fc receptor in the presence of sICAM-1 / CD54 shown in SEQ ID NO: 2. The immunosuppression-sensitive human IgG1 antibody is contacted with biotinylated human CD16a Fc receptor in the presence of sICAM-1 / CD54 shown in SEQ ID NO: 2. Binding of the CD16a Fc receptor by the candidate immunosuppression-resistant IgG1 antibody and the immunosuppression-sensitive IgG1 antibody is determined.

[0016] Another aspect of the present invention is a method for identifying candidates resistant to ICAM-1 / CD54 immunosuppression by screening candidate immunosuppression-resistant human IgG1 antibodies having one to four amino acid substitutions compared to an immunosuppression-sensitive human IgG1 antibody. The immunosuppression-resistant human IgG1 antibody candidate is contacted with biotinylated human C1q protein in the presence of sICAM-1 / CD54 shown in SEQ ID NO: 2. The immunosuppression-sensitive human IgG1 antibody is contacted with biotinylated human C1q protein in the presence of sICAM-1 / CD54 shown in SEQ ID NO: 2. The binding of the immunosuppression-resistant human IgG1 antibody candidate and the immunosuppression-sensitive human IgG1 antibody to the biotinylated human C1q protein receptor is determined.

[0017] Another aspect of the present invention is a method for screening immunosuppression-resistant human IgG1 antibody candidates in antibody-drug conjugate (ADC) format that have one to four amino acid substitutions compared to immunosuppression-sensitive human IgG1 antibodies to identify candidates that are resistant to ICAM-1 / CD54 immunosuppression. The candidate immunosuppression-resistant ADCs are incubated with syngeneic cells of isogenic ICAM-1-expressing cells and non-ICAM-1-expressing cells, and the relative killing of each cell type is determined.

[0018] Yet another aspect of the present invention is a method for identifying tumor-bearing patients who are good candidates for treatment with an antibody-drug conjugate. The expression of ICAM-1 is tested on the tumors of each of a plurality of patients. The testing determines whether the tumors of each of the plurality of patients express ICAM-1. If the tumors do not express ICAM-1, treatment with an IgG1 antibody-drug conjugate is recommended for the patient. The IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression. If the tumors express ICAM-1, it is recommended that the patient not be treated with the IgG1 antibody-drug conjugate. Again, the IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression. Optionally, at least one patient who does not express ICAM-1 is treated with the IgG1 antibody-drug conjugate. The IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression.

[0019] These and other aspects of the present invention will become apparent to those skilled in the art upon reading this specification, and provide the art with methods and compositions for improving antibody-mediated humoral immune responses and the efficacy of antibody-drug conjugates in ICAM-1 immunosuppressive diseases, including cancer and non-cancer diseases. [Brief explanation of the drawings]

[0020] [Figure 1] Identification of ICAM-1 binding to human IgG1. Briefly, 96-well ELISA plates were coated with 1–10 μg / mL of human soluble ICAM-1 (sICAM-1) or human serum albumin (HSA), or without coating, and probed with 1–5 μg / mL of rituximab, pertuzumab, or trastuzumab as previously described (Kline JB, et al. OncoTarget 8:52045–52060, 2017). Each antibody was biotinylated and tested for binding to immobilized sICAM-1 via ELISA. Antibodies were biotinylated using EZ-Link™ Sulfo-NHS-Biotin (ThermoScientific) according to the manufacturer's instructions. Biotinylated antibodies were quantified using Nanodrop™, and signal intensity was verified using an anti-IgG ELISA capture assay to measure the signal intensity of each antibody. All antibodies showed similar signal intensities at similar concentrations (not shown). As shown in Figure 1A, all antibodies were able to significantly bind to sICAM-1, similar to trastuzumab (P = 0.00009). To test the binding of sICAM-1 to immobilized IgG1, 96-well ELISA plates were probed with 5 μg / mL biotinylated sICAM-1 (Sino Biologicals) or biotinylated HSA, both prepared using EZ-Link™ Sulfo-NHS-Biotin as described above. As shown in Figure 1B, sICAM-1 was able to significantly bind to all antibodies, similar to pertuzumab (P = 0.001), but not to HSA. Experiments were performed with a minimum of three wells. Statistical analysis was performed using Student's t-test. [Figure 2A] sICAM-1 inhibits CD16a activation. To test the ability of sICAM-1 to suppress CD16a Fc-γ receptor activation and downstream ADCC, a Jurkat-CD16a-luciferase reporter system was used. In Figure 2A, CD20-positive Daudi cells were used as target cells, and CD16a activation was measured in the presence of increasing amounts of sICAM-1 (0.1–10 μg / mL) using anti-CD20 rituximab antibody according to the manufacturer's protocol. As shown, sICAM-1-treated cultures exhibited a dose-dependent suppression of CD16a activation, with the highest dose demonstrating the most potent inhibitory effect (P = 0.0027). Experiments were performed in a minimum of three wells. Statistical analysis was performed using Student's t-test. Similar analysis of sICAM-1-mediated ADCC inhibition was performed for other antibody panels. As shown in Figure 2B, 10 μg / mL of sICAM-1 was able to inhibit CD16a activation of all antibodies administered at 1 μg / mL. [Figure 2B] See legend to Figure 2A. [Figure 3] Inhibition of CD16a Fc receptor and C1q binding. Suppression of CD16a activation typically results in decreased binding of antigen-bound IgG1 antibodies to the CD16a Fc-γ receptor. To determine whether ICAM-1 binding to IgG1 reduces CD16a Fc-γ receptor binding and complement-mediated C1q protein binding, an ELISA-based assay was performed to monitor their binding in the presence of sICAM-1. Briefly, pertuzumab was coated onto 96-well plates and probed with biotinylated CD16a or C1q protein in the presence of sICAM-1 (10 μg / mL). As shown, binding of CD16a (Figure 3A) and C1q (Figure 3B) proteins was significantly reduced (P = 0.00039 and 0.0033, respectively). Experiments were performed with a minimum of three wells. Statistical analysis was performed using Student's t-test. [Figure 4]Domain mapping of ICAM-1 IgG1 binding. To identify the potential IgG1-binding region of ICAM-1, human IgG1 fragments were generated by papain digestion to obtain F(ab')2 and Fc fragments. Whole IgG1 and fragments were immobilized on 96-well microplates (2.5 μg / mL) and probed with biotinylated sICAM-1 (0.5 μg / mL). As shown in Figure 4A, sICAM-1 specifically binds to the Fc domain (P = 0.00081 compared to HSA). Experiments were performed in a minimum of three wells. Statistical analysis was performed using Student's t-test. Furthermore, secreted recombinant monomeric heavy chain domains (CH2 and CH3) were expressed in 293F cells and tested in a similar manner (Figure 4B). Interestingly, purified Fc and rituximab again showed binding to biotinylated sICAM-1, whereas none of the fragments bound to it. This further localized the ICAM-1 binding domain to the CH2-CH3 region. [Figure 5-1]Deletion and Mutagenesis of the IgG1 Heavy Chain. To further define the region of ICAM-1 that binds to IgG, we generated GST fusion proteins consisting of the Fc domain (from the hinge region to the C-terminus, including the C-terminal Flag tag) and used them in competitive binding assays between ICAM-1 and IgG1 via ELISA. The numbering of the deletions was based on the Kabat residue positions within the IgG1 heavy chain, as summarized in Figure 5A. Constructs 20–24 were also expressed as full-length variants of rituximab. As shown in Figure 5A, GST deletion mutants from Kabat 411 to the C-terminus still significantly bound to sICAM-1 (P = 0.000144, constructs 2 and 3). In contrast, mutants from Kabat 391 to 401 (constructs 4 and 5) lost ICAM-1 binding. Figure 5B shows the results of a representative experiment using constructs 3–5. To further define this region, multiple alanine and / or glycine substitution mutants were generated, both as GST fusions and as full-length rituximab antibodies, for competitive binding of ICAM-1 to wild-type IgG1 heavy chains (Figure 5A, constructs 6-24). As shown in Figure 5C, mutant heavy chains containing alanine and / or glycine substitutions (Kabat substitutions 407-410) completely lost the ability to compete with wild-type IgG1 (constructs 22 and 24), identifying the Kabat 407-410 region as essential for ICAM-1 binding. Assays represent triplicate values, and statistics were determined using Student's t-test. [Figure 5-2] See description of Figure 5-1. [Figure 6] Schematic representation of the ICAM-1 binding region of IgG1 in relation to the CH3 domain and other functional binding sites for CD16a Fc-γ receptor, C1q, and FcRn binding. [Figure 7]Bioassay analysis of the killing effect of saporin-conjugated trastuzumab and pertuzumab antibodies (referred to as ZAP) on HER2+ HCT116 wild-type cells (HCT116-WT) and HCT116 ICAM-1 knockdown cells (HCT116-ICAM1-KO). As shown in the figure, trastuzumab-ZAP and pertuzumab-ZAP significantly killed HCT116-ICAM1-KO cells compared to HCT116-WT cells (P < 0.025). This suggests that ICAM-1 expressed on the membrane surface may negatively affect the internalization and target cytotoxicity of antibodies conjugated to toxins (e.g., ADCs). Assays represent triplicate values, and statistics were determined using Student's t-test. [Figure 8] ELISA analysis of rituximab containing heavy chain CH3 modifications within the sICAM-1 binding domain. Competitive ELISA assays were performed to test sICAM-1 binding to parent rituximab (RTX) and RTX modified at residues 369-372, 374-377, and 407-410 (termed RTX-FARV). As shown, modifications of amino acids within these three regions significantly reduced sICAM-1 binding to rituximab (P < 0.00065). Assays represent triplicate values, and statistics were determined using Student's t-test. [Figure 9] Bioassay analysis of parent rituximab (RTX) and RTX-FARV in the presence of sICAM-1. Jurkat-CD16a reporter cells were tested for activation of CD20+ Daudi target cells using parent rituximab (RTX) or modified RTX (RTX-FARV) in the presence or absence of 10 μg / mL sICAM-1. As shown, both antibodies activated Jurkat-CD16a signaling, but the addition of sICAM-1 significantly inhibited Jurkat-CD16a activation by RTX, whereas it had no effect on RTX-FARV (P = 0.0015). Assays represent triplicate values, and statistics were determined using Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0021] Electronic Sequence Listing Reference The entire contents of the electronic sequence listing (sequencelisting.xml; size: 16,000 bytes, created: August 15, 2022) are incorporated herein by reference.

[0022] Detailed Description of the Invention To determine whether ICAM-1 negatively impacts any of the antibodies that utilize immune effector mechanisms to kill tumors in antibody-based therapies, we examined the physical binding of ICAM-1 to these antibodies. We also examined the humoral immune effector activity of immunosuppressed tumor cells. We found that ICAM-1 binds to IgG1-type antibodies and can negatively impact their humoral immune effector activity. Furthermore, ICAM-1 binding can affect the efficacy of antibody-drug conjugates (ADCs). Cell surface binding of ICAM-1 to antigen-binding ADCs reduces the internalization rate of the ADCs, negatively impacting their efficacy (see Liao, MZ, et al. Clin Pharmacol & Therapeutics 110: 1216-1230, 2021).

[0023] The present inventors have discovered that ICAM-1 protein binds to a specific region in the CH3 domain of human IgG1 antibodies and can suppress their immune effector activity. Here, we demonstrate that modifying this region (the IC1-binding region) can render IgG1 antibodies resistant to ICAM-1-mediated immunosuppression. The use of ICAM-1-binding modified antibodies may improve or enable therapeutic benefit in cancer patients whose tumors overexpress ICAM-1 (see Maruo Y et al. Int J Cancer 100:486-490, 2002; Wu M et al. Path Res Pract 10.1016 / j.prp.2020.153029; Roland CL et al. Surgery 141:705-707, 2007). As specific examples of this approach, ICAM-1 binding-modified rituximab (SEQ ID NOs: 4 and 5), trastuzumab (SEQ ID NO: 6), pertuzumab (SEQ ID NO: 7), cetuximab (SEQ ID NO: 8), and daratumumab (SEQ ID NO: 9) were generated. Modification of the ICAM-1 binding site can be used to engineer the Fc domain of IgG1-type antibodies to generate ICAM-1-resistant antibodies and improve therapeutic efficacy.

[0024] We have shown that modifying Kabat residues 367-425, particularly Kabat residues 369-410 within the CH3 domain (the IC1-binding domain), renders the affected antibodies resistant to ICAM-1-mediated immunosuppression. Modifications can be made, for example, within residues 369-372, 374-377, and / or 407-410 of IgG1-type antibodies. These ICAM-1-resistant (also called IC1-modified) antibodies have the ability to overcome humoral immunosuppression mediated by the ICAM-1 protein. These antibodies can be used to treat cancer as well as other ICAM-1-mediated immunosuppressive diseases. While not wishing to be limited to a particular theory or mechanism of action, Applicants believe that the ICAM-1 protein interacts with residues within and surrounding Kabat residues 369-410 of IgG1-type antibodies to inhibit antibody-mediated humoral immune responses by immune effector cells and / or the complement system. These inhibitions include inhibition of C1q-antibody (classical antibody-complement) complexes and / or inhibition of antibody binding to Fc-γ activating receptors (CD16a, CD32a, CD64a). These occur in immune effector cells, such as, but not limited to, natural killer cells, dendritic cells, monocytic cells, and myeloid cells. These inhibitions lead to downstream suppression of CDC, ADCC, and ADCP. Alterations to other portions of the antibody molecule adjacent to the IC1-binding domain may also affect ICAM-1 binding. Therefore, proximity to the IC1-binding domain may be assessed not only with respect to the primary structure of the antibody, but also with respect to its secondary, tertiary, and quaternary structure.

[0025] Furthermore, recent studies have shown that cell surface protein binding to antibodies can negatively impact the efficacy of antibody-drug conjugates (ADCs) by reducing their internalization (Nicolaides NC, et al. PloS ONE DOI.org / 10.1371 / journal.pone.0285161, 2023). Cell surface expression of ICAM-1 may affect ADCs in a similar manner. Therefore, the use of IC1-modified antibodies may also improve the efficacy of ADC treatments against ICAM-1-expressing tumors.

[0026] The methods described here may be used to develop additional ICAM-1-resistant antibodies targeting tumor-associated antigens, such as CD20, CD38, HER2, or EGFR, to overcome humoral immune suppression and ADC suppression. IC1-modified antibodies that can circumvent the inhibitory effects of ICAM-1 on humoral immune responses and / or ADC suppression may be used in preclinical studies, human trials, and clinical practice.

[0027] In therapeutic applications, IC1-modified antibodies can be administered with or without other standard therapeutic agents, which can be administered before, during, or after administration of the IC1-modified antibody or ADC.

[0028] Compositions formed during the practice of these methods may be useful diagnostically or therapeutically. These compositions may be combinations of proteins with amino acid changes in domains affected by other tumor immunosuppressive proteins, such as CA125 / MUC1 in rituximab (SEQ ID NO: 14) (Grasso L, et al. Oncol Letters 23:2, 2022). Another example is the introduction of IC1 modifications to the Fc domain, including modifications that can alter ADCC, CDC, or FcRn biology, as known to those skilled in the art. Any of the antibody selections described herein can be used to form diagnostic or therapeutic compositions. These antibodies can also be used analytically as laboratory reagents.

[0029] The methods described here for developing compositions by modifying the IC1-binding domain are useful for any antibody whose dynamic conformation changes upon ICAM-1 binding, resulting in suppression of the humoral immune response and / or antibody internalization.

[0030] The "dynamic structure" of an antibody or protein refers to the three-dimensional structure of the antibody at a particular time, which corresponds to the time when the antibody binds to another protein or agent, and the structure of the antibody prior to that time changes to a different structure after that time in response to binding to the other protein or agent.

[0031] Antibody-based approaches continue to be pursued for various cancers as well as inflammatory and infectious diseases, where tumors utilize various pathways to evade host immune defenses. Therefore, it is important to identify agents and regions within antibodies that can be modified to overcome humoral immune suppression and / or affected antibody internalization. These agents or modified antibody compositions and methods enable the selection of lead antibodies that may overcome ICAM-1-induced immune suppression and / or reduced internalization, making them useful for patient screening. For example, patients can be screened to determine whether ICAM-1 expression is elevated. Patients with elevated ICAM-1 expression can be administered IC1-modified antibodies targeting tumor-specific antigens (e.g., rituximab (CD20 antigen), trastuzumab and pertuzumab (HER2 antigen), cetuximab (EGFR antigen), daratumumab (CD38 antigen), etc.) to overcome the inhibitory effects of ICAM-1 on humoral responses and / or internalization, which may affect antibodies in ADC formats.

[0032] The dynamic structure of IgG1 antibodies can be screened in the presence of ICAM-1, which influences the dynamic structure of IgG1 antibodies and suppresses their downstream immune effector function and / or internalization upon binding to target antigens on the cell surface. Figures 1A-1B show an example of screening for ICAM-1 binding, and the following examples and figures demonstrate how such ICAM-1-sensitive antibodies can be modified to resist immunosuppression.

[0033] In one embodiment, ICAM-1-expressing human cancer cells can be used to screen wild-type and IC1-modified antibodies for ICAM-1-mediated immunosuppression.

[0034] In another embodiment, ICAM-1-expressing human cancer cells can be used to screen the antibody internalization rate and / or efficacy of wild-type antibodies and IC1-modified antibody-drug conjugates (ADCs).

[0035] The IC1-modified antibody can contain one or more amino acid changes within or proximal to the IC1-binding domain region (Kabat residues 369-410) of the human IgG1 heavy chain. This proximity can be in the primary structure, or in the secondary, tertiary, or quaternary structure. The amino acid changes can be within Kabat residues 367-425. The IC1-modified antibody can be tested for its ability to overcome ICAM-1-mediated humoral immune suppression of ADCC, ADCP, and CDC, as well as suppressed ADC activity.

[0036] One way to measure the ability of an IC1-modified antibody to overcome humoral immune suppression by ICAM-1 is to test the antibody for direct ICAM-1 binding and / or for its ability to bind to CD16a or C1q proteins in the presence of ICAM-1 by ELISA or other methods familiar to those skilled in the art.

[0037] In another method used to measure the ability of an IC1-modified antibody to overcome ICAM-1 inhibition for an antibody in an ADC format, the antibody is tested for direct ICAM-1 binding and / or target cell internalization rate and / or ADC target cell killing ability in comparison with the IC1-modified format using methods well known to those skilled in the art.

[0038] Functional methods can be used to measure the efficacy of IC1-modified antibodies in overcoming ICAM-1-mediated humoral immunosuppression via ADCC, ADCP, or CDC. ICAM-1 can be produced by target cells or added as a soluble exogenous "agent." The term "efficacy" generally refers to a 10% or greater change in target cell killing via ADCC, ADCP, or CDC when an agent is incubated with the parent and IC1-modified antibodies. This can vary depending on the antibody, but may refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0039] Throughout the specification and claims herein, various terms and terminology relevant to aspects of the accompanying description are used. These terms should be interpreted in accordance with their ordinary meaning in the art unless otherwise expressly stated. Other specifically defined terms should be interpreted in a manner consistent with the definition provided.

[0040] As used in this specification and the appended claims, the singular forms "a," "an," and "the" should be construed to include the plural forms unless the content clearly and specifically dictates otherwise. For example, the term "a cell" may include a combination of two or more cells. The term "probe" may include the parent antibody or IC1 antibody, or an independent antibody against an antigen for monitoring the humoral immune response using any analytical method familiar to those skilled in the art.

[0041] The term "about" is used when referring to amounts, durations, and / or similar quantitative values ​​and is intended to encompass variations of up to ±9% from the specified value. Such variations should be interpreted as appropriate for purposes of practicing the disclosed methods. Unless otherwise expressly indicated, all values ​​expressing quantities of reagents, such as molecular weights, molar concentrations, reaction conditions, and percentages, used in the specification and claims should be understood to be quantified in all instances by the term "about." Thus, unless expressly indicated otherwise, the numerical values ​​set forth in the following specification and recited claims are approximations that may vary depending on the composition of the agent and / or the desired properties of the method sought to be achieved by the present invention. At the very least, and not intended to limit the scope of this application, each numerical value should be evaluated using reported significant digits and ordinary rounding techniques familiar to those skilled in the art.

[0042] The term "antibody" is used broadly to refer to immunoglobulin (Ig) or antibody molecules, including polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs). Monoclonal antibodies include murine, human, humanized, or chimeric mAbs, as well as bispecific antibodies (BSPs), antibody fragments, and antibody-drug conjugates (ADCs). Generally, antibodies are protein or polypeptide chains that bind to a specific antigen. The antigen is the structure specifically recognized by a particular antibody. Canonical antibodies are heterotetramers of glycosylated proteins, consisting of two light chains and two heavy chains held together by a complex of disulfide and hydrogen bonds. The term "disulfide bridges thereof" refers to disulfide bridges contained within the heavy-chain hinge region, which are well known to those skilled in the art. Each heavy chain contains a variable domain (VH), followed by multiple constant domains called CH1, CH2, and CH3, which constitute the Fc domain. Each light chain contains a variable domain (VL) and a constant domain, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the VL of the light chain aligned with the variable domain of the heavy chain. The light chains of any given species can be classified into one of two different types, kappa (κ) or lambda (λ), based on the amino acid sequence within their constant domains.

[0043] Immunoglobulins are classified into classes or isotypes based on the type of Fc domain they contain. Specifically, there are IgA, IgD, IgE, IgG, and IgM, which depend on the sequences contained within the heavy chain constant domain. The IgA and IgG isotypes contain further subclasses, including IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4.

[0044] The VL or VH region of an immunoglobulin consists of a "framework" (FW) region that sandwiches three complementarity-determining regions (CDRs), also known as "antigen-binding sites," based on reported sequence variability (Wu TT, Kabat EA. J Exp Med 132:211-250, 1970). Generally, an antigen-binding site is composed of six CDRs, three of which are located in the variable domain of the heavy chain (CDRH1, CDRH2, CDRH3) and three of which are located in the variable domain of the light chain (CDRL1, CDRL2, CDRL3) (Kabat EA, et al. 5 th Ed. PHS, National Institutes of Health, Bethesda, Md., 1991).

[0045] "Kabat" numbering refers to the system of numbering amino acid residues based on the variable regions of an antibody.

[0046] "Specific binding" or "specifically binds" refers to an antibody or antigen-binding fragment binding to one antigen (including sequences contained in the antibody itself) with higher affinity than to other antigens. Typically, a specific antibody or antigen-binding fragment binds to an antigen with an equilibrium dissociation constant, K D Approximately 5x10 -8 Binds to target antigens at M or less.

[0047] An "antigen" is an entity to which an antibody or antibody fragment specifically binds. This includes binding to an antibody or protein of interest.

[0048] The term "antibody dynamic conformation" refers to conformational changes that may affect the humoral functions of antibodies (e.g., Fc receptor and C1q binding).

[0049] The term "monoclonal antibody (mAb)" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone. This term does not refer to the method by which the antibody is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology, but also includes antibodies produced by recombinant methods.

[0050] "Full-length antibody" refers to an IgG1 type antibody containing the complete variable region and heavy chain domains, typically found in rodent or human serum or produced by recombinant DNA methods.

[0051] "Fab domain" refers to any sequence of an antibody that is N-terminal to the hinge disulfide region of the antibody, as is well known to those skilled in the art.

[0052] "Fc domain" refers to any sequence of an antibody that is C-terminal to the Fab domain of the antibody, including the hinge disulfide region of the antibody, which is well known to those skilled in the art.

[0053] The "affected domain" or "ICAM-1 binding domain" or "IC1 region" refers to the amino acid sequence located within and surrounding Kabat region 369-410 of human IgG1, including, for example, the sequence within Kabat region 407-410 (SEQ ID NO: 3). The affected domain may be contained within the CH3 domain, i.e., Kabat residues 367-425.

[0054] The term "parent antibody" refers to a human IgG1 type antibody composed of a wild-type IgG1 heavy chain sequence.

[0055] The term "IC1-modified antibody" or "IC1 antibody" refers to an antibody having a heavy chain containing one or more amino acid changes within or adjacent to the ICAM-1 binding domain. Substitutions within Kabat region 367-425 (CH3 domain) or Kabat region 369-410, particularly region 407-410, can use any amino acid, including, but not limited to, alanine (ala - A), arginine (arg - R), asparagine (asn - N), aspartic acid (asp - D), cysteine ​​(cys - C), glutamine (gln - Q), glutamic acid (glu - E), glycine (gly - G), histidine (his - H), isoleucine (ile - I), leucine (leu - L), lysine (lys - K), methionine (met - M), phenylalanine (phe - F), proline (pro - P), serine (ser - S), threonine (thr - T), tryptophan (trp - W), tyrosine (tyr - Y), and valine (val - V). One, two, three, or four of the residues within Kabat region 367-425 can be substituted.

[0056] The term "ICAM-1 resistant" refers to an IC1-modified antibody that exhibits better internalization in ADCC, ADCP, CDC and / or ADC formats than the parent antibody.

[0057] The term "agent" refers to any protein or chemical that can block or reduce the immune effector function of an antibody.

[0058] The term "affected antibody" refers to an antibody whose humoral immune function or ability to kill target cells by internalization / antibody drug conjugates is reduced by ICAM-1.

[0059] The term "rituximab" refers to an FDA-approved antibody [FDA Reference ID: 4274293].

[0060] The term "cetuximab" refers to an FDA-approved antibody [FDA Reference ID: 4422941].

[0061] The term "trastuzumab" refers to an FDA-approved antibody [FDA Reference ID: 4090445].

[0062] The term "pertuzumab" refers to an FDA-approved antibody [FDA Reference ID: 3384285].

[0063] The term "daratumumab" refers to the FDA-approved antibody [FDA Reference ID: 4924146].

[0064] The term "CD20" refers to a human cell surface protein expressed by B cells and is the target antigen to which rituximab specifically binds.

[0065] The term "HER2" refers to a human cell surface protein expressed by epithelial cells and is the target antigen to which trastuzumab and pertuzumab specifically bind.

[0066] The term "EGFR" refers to a human cell surface protein expressed by epithelial cells and is the target antigen to which cetuximab specifically binds.

[0067] The term "CD38" refers to a human cell surface protein expressed by lymphoid cells and is the target antigen to which daratumumab specifically binds.

[0068] The term "CA125" refers to the gene product produced by the MUC16 gene (HGNC:15582, OMIM:606154), which exists in soluble and membrane-bound forms and binds to antibodies, affecting their humoral immune functions (Kline JB et al. Oncotarget 8:52045-52060, 2017).

[0069] The term "ICAM-1" refers to the gene product produced by the ICAM-1 / CD54 gene (HGNC:5344, NCBI Reference Sequence:NG_012083.1), which exists in soluble and membrane-bound forms and binds to CD11a / CD18 and CD11b / CD18.

[0070] The terms "cancer," "malignant," "dysregulated," and "tumor" are well known in the art and refer to the presence of cells that have uncontrolled cell growth and morphological characteristics that differ from normal cell types of similar origin. Malignant refers to cancer cells that can cause illness and / or death. As used herein, the terms "cancer" and "tumor" include pre-malignant and malignant types.

[0071] As used herein, the term "soluble" refers to a protein or non-protein agent that is not attached to the plasma membrane of a cell. For example, a soluble agent can be released, secreted, or transported from normal cells or cancer cells into biological fluids, including serum, whole blood, plasma, urine, or cellular microfluids, including tumor cells.

[0072] The "level" of a particular protein agent, including an IC1-modified antibody or ICAM-1, refers to the amount of agent determined by measuring protein levels in vitro or in vivo using methods known in the art, including gel electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation, absorbance spectroscopy, colorimetry, spectrophotometry, flow cytometry, immunodiffusion (single or double), solution-phase analysis, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Förster resonance energy transfer, electrochemiluminescence immunoassay, and the like. In one embodiment, the level of ICAM-1 is determined using probe-based technology.

[0073] The term "humoral immunosuppression" refers to antibodies, antibody fragments, bispecific antibodies (BSPs), or antibody-drug conjugates (ADCs) that are directly bound by ICAM-1 and alter its dynamic conformation. ICAM-1 can exist in membrane-bound or soluble forms.

[0074] The term "bispecific antibody (BSP)" refers to an antibody that can bind to two or more different antigens. A BSP consists of, but is not limited to, at least two full-length antibodies, a full-length antibody and a single-chain antibody, or two single-chain antibodies, each binding to a different antigen or a different epitope on the same antigen.

[0075] The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to an in vitro or in vivo process by which antibodies bind to antigens on the surface of cells and then interact with immune effector cells via sequences within the antibody's Fc domain, causing the release of toxins that kill the bound cells.

[0076] The term "complement-dependent cytotoxicity (CDC)" refers to the in vitro or in vivo process by which antibodies bind to antigens on the cell surface of eukaryotic or prokaryotic cells, then interact with the C1q protein through sequences within the antibody's Fc domain, initiating the classical complement cascade and killing the bound cells.

[0077] The term "opsonization" refers to a process called antibody-dependent intracellular phagocytosis (ADCP), in which antibodies bind to antigens on the cell surface and then interact with immune cells via sequences within their Fc domains, resulting in the immune cells engulfing, consuming, and ultimately killing the antibody-bound cells.

[0078] The terms "native" or "naive" or "canonical" refer to a full-length parent antibody or an IC1-modified antibody without chemical modification.

[0079] The term "antibody drug conjugate (ADC)" refers to an antibody covalently or noncovalently linked to a chemical or biological agent, including radionuclides, nucleic acids, immunotoxins, and small molecular weight compounds. The conjugated agent may be cytotoxic, cytostatic, or diagnostic (emitting a detectable signal via fluorescence, luminescence, radioactivity, or enzymes).

[0080] The term "pharmacokinetics (PK)" refers to the time over which an antibody maintains its steady-state concentration when administered to a subject.

[0081] The term "pharmacodynamics (PD)" refers to the study of the biochemical and physiological effects of antibody-based drugs and their mechanisms of action, including the correlation between their biochemical structure and their effects when administered to a subject.

[0082] The term "pharmacological (PL)" refers to the known effects that antibodies have in controlling or killing disease cells in vitro or in vivo.

[0083] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, and also includes fluids, cells, or tissues present within a subject. Fluids include biological fluids, including liquid solutions that have been in contact with a subject or biological source, such as cell or organoid culture media, urine, saliva, and perfusates.

[0084] The term "control sample" or "control protein" refers to a sample from a healthy subject not suffering from a particular type of cancer, or a clinically or non-clinically relevant control sample containing cells that differ from the parent cells.

[0085] The term "control level" refers to a known or predetermined level (amount) of a protein or non-protein agent used to compare with the level of the same agent used in a sample obtained from a subject or in an in vitro assay.

[0086] As used herein, "difference" refers to the difference between the signal of an antibody under control conditions and the signal when bound to ICAM-1, typically a difference that can be determined by statistical methods familiar to those skilled in the art, and includes a difference of at least 10% compared to the control. Depending on the antibody and probe used, this may also refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0087] The terms "inhibit," "inhibition of," "suppress," and "reduce" refer to decreasing by a statistically measurable amount or preventing altogether.

[0088] The term "functional" indicates that, in an antibody or antibody-containing moiety (e.g., ADC, BSP, etc.) used in accordance with the described methods, the IC1-modified antibody has reduced ICAM-1 binding relative to IgG1-type antibodies and / or is capable of killing target cells more effectively in vitro or in vivo than the parent antibody alone, respectively.

[0089] The term "target cell" refers to a eukaryotic or prokaryotic cell, or population of cells, that expresses an antigen for a particular antibody or antibody-containing moiety.

[0090] The term "pharmacologically acceptable" refers to a substance that can be administered to a patient from a pharmacological and toxicological standpoint and that is manufactured by methods well known to those skilled in the art. This includes agents approved by federal or state regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for animal and human use. The term "pharmacologically compatible ingredient" refers to a pharmacologically acceptable diluent, adjuvant, excipient, or matrix carrier used in administering an anticancer agent. The term "pharmacologically acceptable carrier" refers to a matrix that does not interfere with the effectiveness of the biological activity of the active ingredient and is nontoxic to the host.

[0091] The terms "effective amount" and "therapeutically effective" are used interchangeably and in the context of an amount of a pharmaceutical agent sufficient to improve clinical outcomes when administered to a patient. An effective amount of an agent is administered in an "effective dosing regimen" based on the methods described herein. The term "effective dosing regimen" refers to a combination of an amount of an agent and frequency of administration sufficient to improve clinical outcomes in patients with a particular cancer. "Enhanced efficacy" refers to an improved clinical outcome obtained when administering an agent that has the ability to overcome a disease state more effectively than the parent compound or that can improve the clinical outcome of an effective dosing regimen. As used herein, "effective amount" refers to the amount of IC1-modified antibody required to demonstrate efficacy or differentiation compared to the parent antibody.

[0092] The terms "patient" and "subject" are used interchangeably to refer to humans and other non-human animals (including veterinary subjects) receiving treatment with a therapeutic agent. The term "non-human animal" includes all vertebrate animals. In one embodiment, the subject is a human.

[0093] A "therapeutic agent" is typically substantially free of undesirable contaminants, meaning that the agent is typically at least about 50% w / w (weight / weight) pure and substantially free of interfering proteins and contaminants.

[0094] The term "immune effector cells" refers to cells, including but not limited to natural killer (NK) cells, myeloid cells, monocytic cells, dendritic cells, etc., that can bind to antibody-bound target cells and thereby induce antibody-dependent cellular cytotoxicity (ADCC) or intracellular phagocytosis (ADCP, opsonization). Cells may be purified or present as a mixture in the form of peripheral blood mononuclear cells (PBMCs).

[0095] Inflammatory diseases include autoimmune diseases, rheumatoid arthritis, granulomatosis with polyangiitis, idiopathic thrombocytopenic purpura, pemphigus vulgaris, myasthenia gravis, arteriosclerosis, and Epstein-Barr virus-positive mucocutaneous ulcers.

[0096] Infectious diseases include viral infections, bacterial infections, protozoal infections, and parasitic infections. Viral infections include, but are not limited to, influenza, AIDS, meningitis, pneumonia, herpes, human papillomavirus, respiratory syncytial virus, influenza, Ebola, measles, chickenpox, and shingles. Bacterial infections include, but are not limited to, tuberculosis, whooping cough, Legionnaires' disease, pneumonia, and urinary tract infections. Protozoal infections include, but are not limited to, malaria, giardiasis, Chagas' disease, amebiasis, trichomoniasis, trypanosomiasis, and toxoplasmosis. Parasitic infections include, but are not limited to, filariasis, lice disease, clonorchiasis, pseugotiosis, and cryptosporidiosis.

[0097] The term "dysregulated cells" refers to cells that are considered abnormal compared to their parent cells, including transformed cells, malignant cells, virally infected cells, and cells that exhibit self-regulating, autonomous proliferation.

[0098] The term "test antibody" refers to an antibody for which immune effector activity is to be analyzed.

[0099] The term "humoral response" refers to ADCC, ADCP, and / or CDC activity by a test antibody.

[0100] The term "screening" can refer to testing proteins capable of binding to ICAM-1 in the presence of affected antibodies or antibody-containing moieties (e.g., BSPs, ADCs, single-chain antibodies, antibody fragments, etc.) and monitoring for enhanced biological responses by whole-cell or ADCC-, ADCP-, or CDC-mediated killing. The term may also be used in other contexts in which multiple test elements are analyzed to determine which test elements possess a particular property. Similarly, the term can refer to analyzing patient samples for a particular property, such as elevated ICAM-1 RNA or protein.

[0101] The term "significantly" refers to a statistical result with a P value of less than 0.05 as determined by several programs, including Student's t-test.

[0102] Modified therapeutic antibody compositions and methods for overcoming ICAM-1 / CD54-mediated humoral immune suppression and reduced efficacy of antibody-drug conjugates Binding of ICAM-1 / CD54 to human IgG1 antibodies reduces immune effector activity. Engineered IgG1 antibodies can overcome the ICAM-1 / CD54-mediated suppression of humoral immune responses and the reduced efficacy of antibody-drug conjugates (ADCs) caused by affected parent IgG1 antibodies. In some embodiments, a method for identifying ICAM-1 / CD54-resistant antibodies involves generating IgG1 antibodies containing one or more amino acid changes within or surrounding the affected ICAM-1 / CD54-binding domain (IC1 region) and testing the IC1-modified IgG1 antibodies for significantly improved biological activity when used to mediate ADCC, ADCP, or CDC killing, or improved ADC target cell killing against target cells that specifically express the antigen. In some embodiments, the IgG1 antibody is composed of one amino acid change. In other embodiments, the IgG1 antibody is composed of two or more amino acid changes. Optimal amino acid changes can be determined using functional ADCC, ADCP, CDC, and ADC killing assays in the presence of antibodies with mutated amino acids in or adjacent to the IC1 region. These assays can use primary cells or reporter cells, such as the Jurkat-CD16a ADCC reporter cell line. Examples are described in Example 2 and illustrated in Figures 2 and 8. Identification of ICAM-1-resistant IgG1 antibodies that can significantly overcome ICAM-1 / CD54-mediated humoral immune suppression can also be achieved using molecular assays that monitor antibody binding to the CD16a Fc receptor or C1q protein, as shown in Figure 3, using assays commonly used by practitioners in the field.

[0103] The IC1-modified antibody can have a binding affinity similar to or greater than that of the parent IgG1 antibody.

[0104] Some methods for identifying ICAM-1 / CD54-resistant IgG1 antibodies involve adding the antibody to cultures of target cells expressing the IgG1-specific antigen and ICAM-1, either naturally or recombinantly. Cultures can be monitored using standard CD16a activation, ADCC, ADCP, or CDC killing assays to compare humoral responses in the presence of the ICAM-1-modified and parental antibodies. A significant effect on ADCC and / or CDC function is typically considered to be at least 10%. Depending on the antibody and assay used, a significant effect may be defined as at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%. Target cell lines can be engineered to overexpress ICAM-1 by introducing a human ICAM-1 expression construct or to suppress ICAM-1 expression by shRNA knockdown using shRNA-specific constructs (SEQ ID NOS: 10-13).

[0105] Cancer subjects can be treated with IC1-modified IgG1 antibodies. For example, patients may have cancers that express CD20, including, but not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, or chronic lymphocytic leukemia. Multiple reports have shown that patients with elevated ICAM-1 expression have a poor prognosis in such cancers. Rituximab is the standard treatment for a subset of these cancers. In this case, IC1-modified rituximab antibodies may be desirable. IC1-modified antibodies may be formatted as antibody-drug conjugates or used in their native state.

[0106] Overexpression of ICAM-1 in breast cancer and gastric cancer has been reported to be associated with poor prognosis. The IgG1 antibodies trastuzumab and pertuzumab, both anti-HER2 antibodies, have been approved for the treatment of these cancer indications. In some embodiments, IC1-modified trastuzumab or pertuzumab may be a desirable entity for treating patients with breast cancer or gastric cancer that overexpresses ICAM-1. IC1-modified antibodies may be formatted as antibody-drug conjugates or used in their native state.

[0107] IC1-modified cetuximab, which targets the EGFR protein, has potential therapeutic applications. Cetuximab is approved for the treatment of colorectal cancer and head and neck cancer. Patients with these cancer indications who have elevated ICAM-1 may benefit from IC1-modified cetuximab, making it a desirable entity. IC1-modified antibodies may be formatted as antibody-drug conjugates or may be used in their native state.

[0108] IC1-modified daratumumab, which targets the CD38 protein, has potential therapeutic applications. Daratumumab is approved for the treatment of multiple myeloma. Patients with these cancer indications who have elevated ICAM-1 may benefit from IC1-modified daratumumab, making it a desirable entity. IC1-modified antibodies may be formatted as antibody-drug conjugates or may be used in their native state.

[0109] Patients with CD20-, CD38-, HER2-, or EGFR-positive cancers that overexpress ICAM-1 / CD54 can be treated with IC1-modified rituximab, daratumumab, trastuzumab / pertuzumab, or cetuximab antibodies, respectively, in their native or ADC formats, alone or in combination with standard of care. In some embodiments of the methods described herein for treating a subject with an ICAM-1-overexpressing cancer, the IC1-modified antibody is administered to the subject whose baseline ICAM-1 / CD54 levels are above the normal range. In some embodiments of the methods described herein for treating a subject with an ICAM-1-overexpressing cancer, the method comprises administering an IC1-modified antibody alone. In yet another embodiment, the IC1-modified antibody is administered in combination with chemotherapy, which can be any chemotherapeutic agent considered standard of care for the particular cancer indication at the time the subject is treated. In the treatment methods described herein, ICAM-1 / CD54 expression levels can be determined by any means known in the art, with normal range or above defined as above.

[0110] IC1-modified antibodies may have a single amino acid mutation in the affected domain (Kabat region 369-410). IC1-modified antibodies may have two or more amino acid mutations in the affected domain. IC1-modified antibodies may be administered to patients with tumors that overexpress ICAM-1 / CD54 and express the antigen targeted by the antibody. Representative cancers known to overexpress ICAM-1 / CD54 include Hodgkin's lymphoma and non-Hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, and chronic lymphocytic leukemia. Similar treatments may be applicable to other cancers, including, but not limited to, multiple myeloma, melanoma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, and head and neck cancer.

[0111] This method can be combined with other therapeutic modalities, such as surgery (e.g., debulking surgery), radiation therapy, targeted therapy, chemotherapy, immunotherapy, the use of growth factor inhibitors, or anti-angiogenic factors. The IC1-modified antibody can be administered simultaneously to a patient undergoing surgery, chemotherapy, or radiation therapy. Alternatively, the patient can receive surgery, chemotherapy, or radiation therapy at least one hour before, and up to several months after, administration of the IC1-modified antibody. For example, these treatments can be administered at least one hour, five hours, 12 hours, one day, one week, one month, or three months before, or one hour, five hours, 12 hours, one day, one week, one month, or three months after, administration of the standard of care. In some cases, a therapeutically effective amount of chemotherapy can be administered in combination with an IC1-modified antibody (e.g., rituximab, daratumumab, trastuzumab, pertuzumab, or cetuximab).

[0112] The subject may have already undergone first-line surgical tumor resection or first-line chemotherapy prior to administration of the IC1-modified antibody specific for an antigen expressed by the cancer.

[0113] Therapeutic antibodies containing an IC1-modified heavy chain and CDR sequences capable of directing binding to the CD20 antigen (e.g., SEQ ID NO: 4 or 5); antibodies containing an IC1-modified heavy chain and directed against the HER2 antigen (e.g., SEQ ID NO: 6 or 7); antibodies containing an IC1-modified heavy chain and directed against the EGFR antigen (e.g., SEQ ID NO: 8); and antibodies containing an IC1-modified heavy chain and directed against the CD38 antigen (e.g., SEQ ID NO: 9) may be used, each of which contains one or more modifications within or adjacent to the ICAM-1 / CD54 binding domain (Kabat regions 369-410). The antibodies may be in ADC or native format. Suitable antibodies may be administered to subjects with an antigen-positive disease indication (for which the therapeutic antibody is intended) and whose ICAM-1 / CD54 levels are above the normal range. Treatment may include surgery and standard therapy, as needed.

[0114] The IC1-modified antibody can be administered using a variety of delivery systems, including intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes, as needed. The antibody can be administered systemically or locally, for example, by continuous infusion or bolus injection, through absorption through epithelial or mucosal layers. The antibody is typically administered intravenously.

[0115] The IC1 modified antibody can be administered by injection using a syringe / catheter / or any implantable matrix or device.

[0116] The IC1 modified antibody can be administered in combination with other agents as a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a therapeutic agent and one or more pharmacologically acceptable or compatible ingredients.

[0117] The amount of a therapeutic agent effective for treating or preventing cancer or a non-neoplastic disease can be determined by standard clinical techniques. Furthermore, in vitro assays may be used, if necessary, to identify optimal dosage ranges for the IC1-modified antibody. Effective dosages can be extrapolated from dose-response curves for the IC1-modified antibody derived from in vitro or animal model test systems.

[0118] For example, the toxicity and therapeutic efficacy of IC1-modified antibodies have been demonstrated in cell cultures or experimental animals using LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 This can be expressed as a ratio of . Agents with a large therapeutic index are suitable. If an agent exhibits side effects due to drug toxicity, delivering the agent through a delivery system that targets the site of the affected tissue can minimize potential damage to cells that do not express the antigen, thereby mitigating side effects.

[0119] The nucleic acid vector may be, for example, a plasmid, virus, or subviral particle. Preferably, the nucleic acid has an efficient origin of replication so that it can be maintained in the host cell, although transient expression may be desired in some situations. Expression of an IC1-modified antibody generally requires expression control sequences on the vector and appropriate accessory proteins in the host cell. To produce large amounts of an antibody or antibody fragment, stable cell lines expressing the entire antibody or a fragment are often generated. The antibody fragment may be the entire light or heavy chain. The nucleic acid and vector may be used to generate a cell line expressing the desired antibody. The cell line may be used in a manufacturing process. Alternatively, the antibody-expressing cell line may be delivered directly to the patient in a manner that secretes the antibody. For example, the cell line may be present in a subcutaneous device that produces the protein in situ. Alternatively, the nucleic acid or vector may be delivered to the subject as a form of gene therapy.

[0120] Dosage and administration schedules may vary with effective drug concentration depending on the needs, size, and condition of the subject.

[0121] According to one aspect of the present invention, a method for treating a patient with cancer or an inflammatory disease is provided. An ICAM-1-modified (herein referred to as IC1) human IgG1-type antibody is administered to a patient with cancer or an inflammatory or infectious disease. The administered protein is resistant to ICAM-1 binding and enhances the efficacy of a therapeutic antibody, with or without a modified Fc domain. The IC1-modified antibody may contain one, two, three, or four amino acid modifications; may contain a combination of amino acids that alter the sequence of the wild-type human IgG1 Fc domain within the IC1-binding domain (Kabat residues 369-410); or may lack a portion of the IC1-binding domain. The antibody may have one or more amino acid mutations within or outside the IC1-binding domain (Kabat region 369-410). Any antibody containing a modification in the 367-425 region, regardless of whether it contains a modification in Kabat region 407-410, is referred to herein as an IC1-engineered antibody or IC1-modified antibody.

[0122] Another aspect of the invention involves modifying one or more amino acids within the CH3 domain of human IgG1 (Figure 6), which reduces ICAM-1 binding while retaining the ability to bind to the CD16a Fc receptor and C1q protein.

[0123] IC1-engineered IgG1-type antibodies can be generated that target tumor proteins, including, but not limited to, BCMA, CD19, CD20, CD22, CD30, CTLA-4, CD38, epidermal growth factor, fibroblast growth factor 1, 2, 3, or 4, folate receptor alpha, HER2, mesothelin, PD-1, and PD-L1. All of these tumor proteins are known in the art.

[0124] It may be possible to treat patients with cancer or inflammatory diseases in which levels of sICAM-1 or membrane-bound ICAM-1 are elevated compared to healthy human populations. The treatment involves an IC1-modified antibody that targets an antigen specific to diseased cells. The agent is typically administered daily, weekly, every three weeks, or monthly.

[0125] Patients with non-Hodgkin's lymphoma (NHL) can be treated with IC1-modified rituximab, which contains a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 4), or an antibody containing the heavy chain of CA125-resistant rituximab (Grasso L et al. Oncol Letters 23:2, 2022) (SEQ ID NO: 5). The IC1-modified rituximab antibody can be administered to patients alone or in combination with other chemotherapeutic agents. The antibody binds to CD20-positive target cells and induces a humoral immune response, such as ADCC, ADCP, or CDC, resulting in the killing of the bound cells. The antibody can be in ADC or native format.

[0126] Patients with HER2-positive breast cancer, gastric cancer, or head and neck cancer can be treated with IC1-modified trastuzumab, which contains a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 6). The IC1-modified trastuzumab antibody can be administered to patients alone or in combination with other chemotherapy agents. The antibody binds to HER2-positive target cells and induces a humoral immune response, such as ADCC, ADCP, or CDC, resulting in the killing of the bound cells. The antibody can be in ADC or native format.

[0127] Patients with HER2-positive breast cancer, gastric cancer, or head and neck cancer can be treated with IC1-modified pertuzumab, which contains a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 7). The IC1-modified pertuzumab antibody can be administered to patients alone or in combination with other chemotherapeutic agents. The antibody binds to HER2-positive target cells and induces a humoral immune response, such as ADCC, ADCP, or CDC, which kills the bound cells. The antibody can be in ADC or native format.

[0128] Patients with colorectal cancer or head and neck cancer can be treated with IC1-modified cetuximab, which contains a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 8). The IC1-modified cetuximab antibody can be administered to patients alone or in combination with other chemotherapeutic agents. The antibody binds to EGFR-positive target cells and induces a humoral immune response, such as ADCC, ADCP, or CDC, resulting in the killing of the bound cells. The antibody can be in ADC or native format.

[0129] Patients with multiple myeloma can be treated with IC1-modified daratumumab, which contains a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 9). The IC1-modified daratumumab antibody is administered to patients alone or in combination with other chemotherapeutic agents. The antibody binds to CD38-positive target cells and induces a humoral immune response, such as ADCC, ADCP, or CDC, resulting in the killing of the bound cells. The antibody can be in ADC or native format.

[0130] In the following examples, we demonstrate that ICAM-1 (SEQ ID NOS: 1 and 2) can directly bind to IgG1-type immunoglobulins and inhibit their immune effector functions. Furthermore, we have identified a specific ICAM-1-binding region (referred to as the "IC1" binding region) on the human IgG1 Fc domain. By modifying this region, immunoglobulins that are sensitive to immunosuppression by soluble ICAM-1 (sICAM-1) (SEQ ID NOS: 2) can be made resistant to it. Furthermore, ICAM-1-resistant antibodies can also be generated by modifying residues located in close proximity to the IC1 binding region.

[0131] Binding of IgG1 to membrane-bound ICAM-1 (mICAM-1) (SEQ ID NO: 1) may be involved in inflammatory diseases. IC1 modifications can be made to wild-type IgG1 Fc domains or IgG1 Fc domains with additional modifications to enhance or decrease ADCC, ADCP, and / or CDC activity (Natsume A, et al. Drug Design, Devel Therapy 3:7-16, 2009; Saunder KO. Front Immunol 10:1296-1316, 2019). Furthermore, modifying the Fc domain to enhance neonatal Fc receptor (FcRn) binding can improve serum half-life (Dall'Acqua WF et al. J Biol Chem 281:23514-23524, 2006). Additionally, these modifications can be incorporated into various antibody formats, such as bispecific antibodies (BSPs) and antibody-drug conjugates (ADCs), to improve therapeutic activity. [Example]

[0132] Example 1 - Screening for proteins that inhibit the humoral immune effector function of antibodies Recent studies have demonstrated that some tumor-produced proteins suppress antibody-mediated killing via ADCC, ADCP, and / or CDC (Kline JB, et al. J Clin Oncol 5:15, 2018; Kline JB et al. Eur J Immunol. 48:1872-1882, 2018; Grasso L et al. Oncol Letters 23:2, 2022). Furthermore, antibodies conjugated to these proteins have been shown to be adversely affected in antibody-drug conjugate formats (Nicolaides NC, et al. PloS ONE DOI.org / 10.1371 / journal.pone.0285161, 2023). This immunosuppressive activity is thought to arise through direct binding to specific regions of the affected antibodies. To identify additional humoral immunosuppressive proteins, we used molecular biology assays to test candidate proteins reported by other researchers to be produced by various types of tumors and associated with poor prognosis for their potential as antibody immunosuppressants. ICAM-1 protein has been associated with various cancers by several research groups and has been reported to correlate with poor prognosis. To assess the impact of antibodies on immune effector activity, we evaluated the antibody binding ability of soluble ICAM-1 (sICAM-1). The screening assay employed a 96-well plate ELISA. Wells were coated with sICAM-1 protein (Sino Biologicals, SEQ ID NO: 2), and binding was measured using a biotinylated human antibody as a probe. As shown in Figure 1A, all IgG1 antibodies tested bound to wells containing sICAM-1, in contrast to wells coated with human serum albumin (HSA) protein, used as a negative control. Next, ICAM-1 / IgG1 binding was tested using immobilized IgG1 or IgM antibodies and biotinylated sICAM-1 or HSA. As shown in Figure 1B, when wells containing IgG1-type antibodies were probed with sICAM-1, strong binding was observed in contrast to wells containing a control protein (IgM / HSA), demonstrating the ability of sICAM-1 to directly bind to IgG1-type antibodies.

[0133] Binding of a protein to an IgG1 antibody may or may not affect its ability to suppress immune effector activity. To examine the effect of sICAM-1 binding to an IgG1 antibody on immune effector activity, we employed a biological assay monitoring ADCC activity. It has been reported that membrane-bound ICAM-1 promotes ADCC by natural killer (NK) cells via LFA binding, and disruption of this interaction has been shown to reduce ADCC activity against target cells (Cooley S et al. Exp Hematol 27:1533-1541, 1999; Sanchez-Martinez D et al. Theranostics 8:3856-3869, 2018). To circumvent the potential complications of the ICAM-1 / LFA pathway when interpreting the effect of sICAM-1 on IgG1-mediated ADCC, we employed the Jurkat-CD16a-luciferase (Jurkat-CD16a) system (Promega) to monitor CD16a activation (a prerequisite for effector cell-mediated ADCC activity). In this assay, CD20-positive Daudi cells were used as target cells, and anti-CD20 rituximab (SEQ ID NO: 4) was used as the targeting IgG1 antibody. Briefly, 2 × 10 4 Target cells were seeded overnight in triplicate in black opaque 96-well plates in R1 assay buffer (RPMI + L-glutamine + 1% ultra-low Ig serum) (Gibco). The next day, 1 × 10 5Jurkat-CD16a effector cells were added to wells in R1 assay buffer at a 5:1 effector-to-target ratio. Rituximab at 2.5 μg / mL and sICAM-1 (ranging from 0 to 10 μg / mL) were added, and the plate was incubated at 37°C and 5% CO2 for 16 hours. After incubation, the microwell plate was equilibrated at room temperature for 30 minutes, and Jurkat-CD16a activation was measured using BIO-GLO luciferase reagent according to the manufacturer's protocol (Promega). CD16a activation was quantified using a Varioskan LUX plate reader (ThermoFisher). As shown in Figure 2A, sICAM-1 demonstrated a significant dose-response effect (P<0.003) in suppressing rituximab-induced CD16a activation on Daudi cells. To confirm the efficacy of sICAM-1 against other IgG1-type antibodies, we performed a similar Jurkat CD16a activation assay using trastuzumab, pertuzumab, cetuximab, and rituximab (repeat control) at 1 μg / mL in the presence of 10 μg / mL sICAM-1. For trastuzumab and pertuzumab, the HER2-expressing human SK-BR-3 breast cancer cell line was used as the target cell. For cetuximab, the EGFR-expressing A431 epidermoid carcinoma cell line was used as the target cell. As shown in Figure 2B, sICAM-1 was able to inhibit CD16a activation with all four antibodies (P < 0.008). In toxicity assays of sICAM-1 against effector and target cells, co-culture at 10 μg / mL was well tolerated, eliminating the possibility of artifactual effects in the assay.

[0134] To demonstrate the effect of ICAM-1 on suppressing the immune effector activity of IgG1, we generated isogenic ICAM-1 knockdown cells using the ICAM-1-expressing HCT116 cell line. Briefly, these cells were grown in 6-well plates at 7.5 × 10 cells per well in 3 mL of complete RPMI (RPMI-1640 supplemented with 7.5% fetal bovine serum and 1% L-glutamine). 5Cells were seeded and cultured overnight at 37°C under 5% CO2. The next day, shRNA constructs (SEQ ID NOs: 10–13) targeting the human ICAM-1 sequence (Origene TG312270) were transfected using Lipofectamine 3000 reagent (Sigma) according to the manufacturer's instructions. Four independent sequences and a scrambled sequence negative control were used. After transfection, cells with stable integration of the expression construct were selected using puromycin selection. After selection, cultures were cloned into single cells in 96-well plates, and protein lysate samples were collected and analyzed for loss of ICAM-1 expression by Western blotting using anti-hICAM-1 rabbit polyclonal antibody (Sino Biologicals) diluted 1:1000. Knockdown clones were expanded for banking and tested for reduction in IgG1 immune effector activity to confirm the immunosuppressive effects of sICAM-1 and membrane-bound ICAM-1 compared to isogenic parental cells.

[0135] Furthermore, we used cells to monitor the inhibitory effect of membrane-bound ICAM-1 on IgG1 antibody-drug conjugate (ADC) activity, potentially due to the suppression of internalization via the physical interaction between the IgG1 Fc domain and ICAM-1. Previous studies on antibodies in ADC format have shown that antibody-interacting proteins such as MUC16 / CA125 interfere with ADC internalization and reduce target cytotoxicity (Nicolaides NC et al. (PLOS ONE May 17;18(5):e0285161, 2023)). To determine the effect of ICAM-1 on antibodies in ADC format, the ZAP streptavidin-saporin antibody conjugation system was employed according to the manufacturer's protocol (Advanced Targeting Systems). ZAP conjugates saporin toxin to biotinylated antibodies, and when the antibody-saporin complex is internalized into antigen-expressing target cells, saporin is released and exerts toxicity by ribosome inhibition (Polito, L, et al. Toxins 5:1698-1722, Briefly, trastuzumab and pertuzumab antibodies were biotinylated using EZ-link biotinylation reagent according to the manufacturer's protocol (ThermoScientific). Next, 100 nM biotinylated antibody and 100 nM streptavidin-saporin were added to RPMI 7.5% FBS growth medium. 100 μL of the solution was added to each well of HCT116 wild-type (HCT116-WT) and HCT-116-ICAM-1 knockdown cells seeded at 5,000 cells / well in a clear 96-well microplate. Wells containing 100 nM ZAP alone and no ZAP antibody were used as negative controls. After 5 days of incubation at 37°C and 5% CO2, the killing effects of trastuzumab-ZAP and pertuzumab-ZAP were analyzed using crystal violet blue and measured using a Varioskan plate reader. Quantification was performed in nm.As shown in Figure 7, trastuzumab-ZAP and pertuzumab-ZAP demonstrated significant killing effects against HCT116-ICAM-1 knockdown cells (HCT116-ICAM1-KO) compared with HCT116 wild-type (HCT116-WT) cells (P<0.025). The wild-type and knockout cell lines expressed similar levels of HER2 receptors. All experiments were performed in triplicate, and statistical analysis was performed using Student's t-test. These data support the idea that ICAM-1 binding to antibodies in ADC format can inhibit their internalization activity and target cytotoxicity. Creating antibodies with deleted or modified ICAM-1 binding (IC1) domains is a useful approach for obtaining optimized ADCs. Alternatively, patient tumors can be screened for ICAM-1 membrane expression; tumors lacking ICAM-1 expression may be suitable for ADC-based therapy.

[0136] The effects of tumor immunosuppressive proteins on the immune effector activity of antibodies have previously been shown to be due to reduced CD16a and / or C1q binding to the Fc IgG domain (Kline JB, et al. J Clin Oncol 5:15, 2018; Kline JB et al. Eur J Immunol. 48:1872-1882, 2018). To assess the ability of sICAM-1 to inhibit the binding of these proteins to the IgG1 Fc domain, ELISA assays were performed as previously described (Kline JB, et al. Oncotarget 8:52045-52060, 2017; Kline JB et al. Eur J Immunol. 48:1872-1882, 2018). Briefly, 96-well plates were coated with 1 μg / mL pertuzumab or HSA (used as a negative control) in 0.05 M carbonate buffer overnight at 4°C. The next day, wells were washed with pH 7.2 phosphate buffer (PB) and blocked with 5% BSA in PB for 1 hour. They were then washed three times with PB. Next, wells were probed with 2.5 μg / mL biotinylated soluble human CD16a (Sino Biologicals) or 1 μg / mL biotinylated C1q (Sigma) at room temperature for 1 hour, followed by secondary probing with streptavidin-horseradish peroxidase (HRP). Afterwards, wells were washed three times with PB, and binding was measured using TMB chromogenic substrate. The reaction was stopped with 0.1N H2SO4, and the optical density (A) at 450 nm was quantified using a Varioskan plate reader. As shown in Figures 3A and 3B, sICAM-1 significantly inhibited CD16a Fc receptor binding (P = 0.0039) and C1q binding (P = 0.0033), respectively. Similar results were observed with other antibodies tested using the same method. These data confirm the inhibitory effect of physical binding of ICAM-1 on antibody humoral immune effector activity.

[0137] Example 2 - Identification of key residues required for ICAM-1 binding and generation of ICAM-1-resistant IgG1 antibodies To identify the binding site or domain of ICAM-1, we employed IgG1 fragmentation followed by site-directed mutagenesis. First, fragment domain analysis was performed. Human IgG1 antibodies were digested with papain to generate F(ab')2 and Fc fragments, which were then purified using Protein A. F(ab')2 was isolated from the Protein A flow-through, and the Fc fragment was captured by Protein A and eluted as previously described (Grasso L et al. Oncol Letters 23:2, 2022). The isolated fragments were quantified using a Nanodrop (ThermoFisher), and equal amounts were used in an ELISA format to identify the sICAM-1 binding site. Briefly, plates were coated with 100 μL of full-length IgG1 antibody or fragment at 2.5 μg / mL in 0.05 M carbonate buffer for 1 hour at room temperature. Plates were washed with phosphate-buffered saline (PBS) + 0.1% Tween-20 (PBS-T) and then blocked with 200 μL of 1% BSA in PBS. After washing wells with PBS-T, 0.5 μg / mL biotinylated sICAM-1 was dissolved in 1% BSA in PBS and probed in triplicate for 1 hour at room temperature on a shaking platform. After washing the plate three times with PBS-T, secondary probing was performed with 333 ng / mL streptavidin-HRP in 1% BSA in PBS on a shaking platform for 1 hour at room temperature. After washing the plate three times with PBS-T, quantification was performed using TMB chromogenic substrate (Pierce). The reaction was stopped with 0.1N H2SO4, and A450 was quantified using a Varioskan plate reader. As shown in Figures 4A-4B, sICAM-1 binds to the Fc domain (P = 0.00081 compared to HSA).

[0138] To further define the sICAM-1 binding site within the Fc domain, deletion mutagenesis and ELISA competition assays were performed. A cDNA fusion construct containing an Ig leader sequence, a GST protein, and an IgG1 fragment consisting of the hinge to CH3 domain of IgG1, followed by a C-terminal Flag tag, was synthesized (Genscript) and subcloned into a CMV promoter-driven eukaryotic expression plasmid. For deletion mutagenesis, fragments were amplified from the full-length Fc construct using a 5' primer encoding a HindIII and Kozak sequence and a 3' primer encoding an EcoRI and Flag tag. Each fragment was isolated and subcloned into the expression plasmid. For site-directed mutagenesis, the 5' and 3' regions were PCR amplified using overlapping primers encoding the codon changes, purified, and then used in a second-stage PCR using flanking primers. The resulting fragments were cloned into the expression plasmid. All constructs were sequenced to confirm accuracy. Culture supernatants were analyzed by Western blotting using anti-FLAG antibody to ensure appropriate molecular weight and quantification. The constructs were stably transfected into 293F cells, and culture supernatants were collected and tested for the ability of the mutant proteins to compete with WT IgG1 by ELISA assay.

[0139] For the ELISA competition assay, 96-well plates were coated with 2.5 μg / mL rituximab in 0.05 M carbonate buffer for 1 hour at room temperature. The plates were blocked as described above. After blocking, culture supernatants containing various expression fragments were added in triplicate with 0.5 μg / mL biotinylated sICAM-1 or 1.5 μg / mL His-tagged full-length sICAM-1 and probed for 1 hour at room temperature under shaking conditions. After washing the plates three times with PBS-T, secondary probing was performed for 1 hour on a shaking platform with 333 ng / mL streptavidin-HRP for detection of biotinylated ICAM-1 or anti-His-HRP (Sino Biologicals) diluted 1:3,000 in PBS containing 1% BSA. The plates were then washed three times with PBS-T, and TMB substrate was added for colorimetric quantification. The reaction was stopped with 0.1 N H2SO4, and A450 was quantified using a Varioskan plate reader. Mutants that lost sICAM-1 binding were unable to compete with full-length sICAM-1 binding to rituximab coated on plates, indicating that they contained critical residues. As summarized in Figure 5A and demonstrated in Figures 5B and 5C, this domain and amino acids are located in the region of Kabat residues 407-410. Further analysis using amino acid substitutions within this region confirmed that these sequences are critical for ICAM-1 binding, as determined by the competitive ELISA screen described above. As shown in Figure 8, additional amino acid substitution analysis unexpectedly revealed that amino acid residues adjacent to these motifs (e.g., Kabat residues 369-372 and Kabat residues 374-377) could also negatively affect ICAM-1 binding to IgG1, suggesting that modifications within Kabat region 369-410 could be used to generate ICAM-1-resistant antibodies and antibody-drug conjugates.

[0140] Example 3 - Design of ICAM-1-resistant IgG1 antibodies to enhance immune effector activity to maximize tumor cell killing efficacy of naked IgG1-based therapies and antibody-drug conjugates (ADCs) Based on the results obtained in Example 2, the development of an IgG1 Fc domain resistant to ICAM-1 binding allows the development of ICAM-1-resistant IgG1 antibodies to maximize immune effector activity and enhance the efficacy of antibody-drug conjugates (ADCs). As described above, the use of isogenic cells in which the ICAM-1 protein is knocked down or knocked out allows the screening of IgG1 mutations within or adjacent to the IC1 region. Furthermore, the ability to create IgG1 heavy chain cassettes with modifications as shown in Figure 6, or cassettes with one, two, three, or four amino acid substitutions within SEQ ID NO: 3 (or adjacent positions, e.g., Kabat 369-372 or Kabat 374-377), allows the development of IgG1 molecules with improved immune effector activity. Additionally, it also allows the development of ADCs that are not hindered by reduced ICAM-1 binding or internalization rates, parameters essential for maximal ADC target cell killing.

[0141] IC1-modified IgG1 is highly useful in the presence of ICAM-1-positive cancers, including, but not limited to, lymphoma, multiple myeloma, breast cancer, gastric cancer, head and neck cancer, and colorectal cancer. Patients are first screened to determine their ICAM-1 status; patients expressing ICAM-1 levels above 5% of the normal range by immunohistochemistry or serum ELISA capable of monitoring sICAM-1 can benefit from administration of IC1-modified antibodies. IC1-modified antibodies include, but are not limited to, IC1-modified rituximab (SEQ ID NOs: 4 and 5), daratumumab (SEQ ID NO: 9), trastuzumab (SEQ ID NO: 6), pertuzumab (SEQ ID NO: 7), and cetuximab (SEQ ID NO: 8). As shown in Figure 9, IC1-modified rituximab (containing four modified amino acids within Kabat 407-410, designated RTX-FARV) exhibited significantly enhanced ADCC activity in the presence of sICAM-1 compared to parent rituximab (RTX) (P=0.0015) using the Jurkat-CD16a reporter cell line and Daudi target cell assay described in Example 1. These results support the use of the improved antibody in the treatment of diseases associated with overexpression of the immunosuppressive ICAM-1 protein.

[0142] All references cited herein are expressly incorporated herein.

[0143] Amino acid sequence TIFF2025528832000002.tif239160TIFF2025528832000003.tif220153TIFF2025528832000004.tif35156

Claims

1. An immunosuppression-resistant human IgG1 antibody comprising 1 to 4 amino acid substitutions relative to an immunosuppression-sensitive human IgG1 antibody, wherein the immunosuppression-sensitive human IgG1 antibody does not have the 1 to 4 amino acid substitutions, and immunosuppression of the immunosuppression-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

2. The immunosuppression-resistant human IgG1 antibody of claim 1, which is a full-length antibody.

3. The immunosuppression-resistant human IgG1 antibody of claim 1, which is an antibody-drug conjugate.

4. The immunosuppression-resistant human IgG1 antibody according to claim 1, wherein the ICAM-1 / CD54 is membrane-bound ICAM-1 / CD54 as shown in SEQ ID NO:

1.

5. The immunosuppression-resistant human IgG1 antibody according to claim 1, wherein the ICAM-1 / CD54 is a soluble ICAM-1 / CD54 shown in SEQ ID NO:

2.

6. The immunosuppression-resistant human IgG1 antibody of claim 1, wherein the 1 to 4 amino acid substitutions are within Kabat residues 367 to 425.

7. The immunosuppression-resistant human IgG1 antibody of claim 1, wherein the 1 to 4 amino acid substitutions are within Kabat residues 369 to 410.

8. The immunosuppression-resistant human IgG1 antibody of claim 6, wherein the 1 to 4 amino acid substitutions are within the motif YSKL (SEQ ID NO: 3) located in the heavy chain of the immunosuppression-resistant human IgG1 antibody.

9. The immunosuppression-resistant human IgG1 antibody of claim 6, comprising an IgG1 light chain and an IgG1 heavy chain.

10. The immunosuppression-resistant human IgG1 antibody of claim 1, conjugated to a drug.

11. A polynucleotide encoding the immunosuppression-resistant human IgG1 antibody of claim 1.

12. A nucleic acid vector encoding the immunosuppression-resistant human IgG1 antibody of claim 1.

13. A stable cell line comprising the nucleic acid vector of claim 12, which expresses an immunosuppression-resistant human IgG1 antibody.

14. 1. A method of treating a patient having a disease, comprising: the patient expresses elevated levels of ICAM-1 / CD54 compared to a healthy human population; The method comprises administering to the patient the immunosuppression-resistant human IgG1 antibody of claim 1. method.

15. 15. The method of claim 14, wherein the disease is cancer.

16. 15. The method of claim 14, wherein the disease is an inflammatory disease.

17. 16. The method of claim 15, wherein the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, non-small cell lung cancer, breast cancer, colorectal cancer, gastric cancer, and head and neck cancer.

18. 1. A method of treating a patient with cancer or an inflammatory disease, comprising: administering to said cancer patient or inflammatory disease patient a full-length human IgG1 antibody comprising a heavy chain having one to four amino acid substitutions in the motif YSKL (SEQ ID NO: 3) located in the heavy chain of said full-length human IgG1 antibody. A method comprising:

19. 19. The method of claim 18, wherein the full length human IgG1 antibody comprises the light chain of rituximab in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

4.

20. 20. The method of claim 19, wherein the full length human IgG1 antibody comprises the light chain of rituximab-N109D in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

5.

21. 19. The method of claim 18, wherein the full length human IgG1 antibody comprises the light chain of trastuzumab in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

6.

22. 19. The method of claim 18, wherein the full length human IgG1 antibody comprises the light chain of pertuzumab in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

7.

23. 19. The method of claim 18, wherein the full length human IgG1 antibody comprises the light chain of cetuximab in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

8.

24. 19. The method of claim 18, wherein the full length human IgG1 antibody comprises the light chain of daratumumab in combination with the immunosuppression-resistant heavy chain as set forth in SEQ ID NO:

9.

25. 25. The method of claim 18, 19, 20, or 24, wherein the patient is a cancer patient, and the patient has a cancer selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, and multiple myeloma.

26. 23. The method of claim 21 or 22, wherein the patient is a cancer patient, and the patient has a cancer selected from the group consisting of breast cancer, gastric cancer, and head and neck cancer.

27. 24. The method of claim 23, wherein the patient is a cancer patient, and the patient has a cancer selected from the group consisting of colorectal cancer and head and neck cancer.

28. 1. A method of screening candidate antibodies to identify antibodies resistant to ICAM-1 / CD54 immunosuppression, comprising: (a) contacting a first human cancer cell line that expresses an antigen with a candidate IgG1 type antibody that specifically binds to the antigen, wherein the contacting is performed in the presence of ICAM-1 / CD54 (SEQ ID NO: 1); (b) contacting a second human cancer cell line that expresses the antigen with a candidate IgG1 type antibody that specifically binds to the antigen, wherein the contacting is performed in the absence of ICAM-1 / CD54 (SEQ ID NO: 1); and (c) determining antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) of the first and second human cancer cell lines stimulated with the candidate antibodies in steps (a) and (b). A method comprising:

29. 29. The method of claim 28, wherein the first human cancer cell line used in step (a) expresses ICAM-1 / CD54, and the second human cancer cell line used in step (b) is an isogenic cell line with reduced or absent expression of ICAM-1 / CD54.

30. 29. The method of claim 28, wherein the first human cancer cell line used in step (a) and the second human cancer cell line used in step (b) are the same cell line.

31. 30. The method of claim 29, wherein the second isogenic human cancer cell line with reduced or deleted expression of ICAM-1 / CD54 comprises shRNAs 1-4 (SEQ ID NOs: 10-13).

32. The method of claim 30, wherein the first and second human cancer cell lines do not express ICAM-1 / CD54, and the presence of ICAM-1 in step (a) is due to the addition of exogenous soluble ICAM-1.

33. 1. A method for screening candidate immunosuppression-resistant human IgG1 antibodies having 1 to 4 amino acid substitutions relative to an immunosuppression-sensitive human IgG1 antibody to identify candidates resistant to ICAM-1 / CD54 immunosuppression, comprising: (a) contacting a candidate immunosuppression-resistant human IgG1 antibody with a biotinylated human CD16a Fc receptor, wherein the contacting is performed in the presence of sICAM-1 / CD54 as set forth in SEQ ID NO:2; (b) contacting an immunosuppression-sensitive human IgG1 antibody with a biotinylated human CD16a Fc receptor, wherein the contacting is performed in the presence of sICAM-1 / CD54 as set forth in SEQ ID NO: 2; and (c) determining the binding of CD16a Fc receptor by the antibodies in steps (a) and (b). A method comprising:

34. 1. A method for screening candidate immunosuppression-resistant human IgG1 antibodies having 1 to 4 amino acid substitutions relative to an immunosuppression-sensitive human IgG1 antibody to identify candidates resistant to ICAM-1 / CD54 immunosuppression, comprising: (a) contacting a candidate immunosuppression-resistant human IgG1 antibody with biotinylated human C1q protein, wherein the contacting is performed in the presence of sICAM-1 / CD54 as set forth in SEQ ID NO:2; (b) contacting an immunosuppression-sensitive human IgG1 antibody with biotinylated human C1q protein, wherein the contacting is performed in the presence of sICAM-1 / CD54 as set forth in SEQ ID NO: 2; and (c) determining the binding of biotinylated human C1q protein by the antibodies in steps (a) and (b). A method comprising:

35. 1. A method for screening candidate immunosuppression-resistant human IgG1 antibodies in an antibody-drug conjugate format having 1 to 4 amino acid substitutions relative to an immunosuppression-sensitive human IgG1 antibody to identify candidates that are resistant to ICAM-1 / CD54 immunosuppression, comprising: (a) contacting a candidate immunosuppression-resistant human IgG1 antibody with a first target cell that expresses ICAM-1 / CD54 and expresses a target antigen, wherein the first target cell is an isogenic cell; (b) contacting the candidate immunosuppression-resistant human IgG1 antibody with a second target cell that does not express ICAM-1 / CD54 and expresses the target antigen, wherein the second target cell is an isogenic cell; and (c) comparing the amount of cell death caused by the candidate immunosuppression-resistant human IgG1 antibody in steps (a) and (b). A method comprising:

36. 1. A method for identifying a tumor-bearing patient who is an eligible candidate for treatment with an antibody drug conjugate, comprising: testing a tumor from each of a plurality of patients to determine whether the tumor from each of the plurality of patients expresses ICAM-1; If the tumor does not express ICAM-1, it is recommended to treat the patient with an IgG1 antibody-drug conjugate, and the IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression; If the tumor expresses ICAM-1, it is recommended not to treat the patient with IgG1 antibody-drug conjugates, as the IgG1 antibody of the antibody-drug conjugates is sensitive to immunosuppression; determining; and Optionally, treating at least one patient who does not express ICAM-1 with an IgG1 antibody-drug conjugate, wherein the IgG1 antibody of the antibody-drug conjugate is susceptible to immunosuppression. A method comprising:

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