Fc engineered monoclonal antibody against tumor immunosuppression ICAM-1 / CD54

Abstract

The ICAM-1 / CD54 protein has been found to be able to inhibit antibody-mediated humoral immunity by binding directly to the CH3 domain of the IgG1 heavy chain. The present invention relates to antibody drug conjugates that bind by reducing the binding of IgG1 to the Fc receptor and C1q complement promoter protein on effector cells, which inhibits the immune effector activity of the antibody, and that it is possible to reduce the activity of antibody drug conjugates by slowing the internalization of target cells. By modifying the CH3 region, engineered antibodies can be generated, which reduce or eliminate the binding of ICAM-1 / CD54, thereby obtaining improved immune effector activity and improved killing effect of the antibody drug conjugate in the presence of ICAM-1 / CD54. These antibodies are useful in the treatment of patients suffering from cancer and inflammation and infectious diseases with high expression of ICAM-1 / CD54.

Description

Technical Field of the Invention

[0001] The present invention relates to the fields of humoral immunity and humoral immune oncology. Specifically, it relates to methods and pharmaceutical formulations capable of overcoming the immunosuppressive effects of the ICAM-1 / CD54 protein, which can enhance the efficacy of antibody-based therapies in inhibiting cancer growth and other humoral immune-suppressive diseases. Reference to the Electronic Sequence Listing

[0002] The content of the electronic sequence listing (008966.00037sequence listing.xml; file size: 15 kb; Creation date: July 31, 2023) is hereby incorporated by reference in its entirety. Background of the Invention

[0003] Humoral immunity is one of the main mechanisms used by vertebrate hosts to monitor and defend against dysregulated and transformed host cells. In cancer biology, immune checkpoint inhibitors that can overcome suppressed cell-mediated immunity have shown a powerful effect of activated CD8+ T cells killing tumor subsets (Hodi FS et al., New England Journal of Medicine 363:711-723, 2010). It has been reported that several commercially approved therapeutic antibodies exhibit their tumor-killing effects through humoral-mediated antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015; Ruck T et al., International Journal of Molecular Sciences 16:16414-16439, 2015; Pelaia C et al., Biomed Research Int 4839230:1-9, 2018). The latest translational research results indicate that tumors produce factors that can inhibit the humoral immune pathway, thereby inhibiting the tumor-killing effect through ADCC, ADCP, and CDC (Vergote I et al., J Clin Oncol 34:2271-2278; Kline JB et al., J Clin Oncol 5:15, 2018; Wang W et al., Cytogenet Genome Res 152:169-179, 2017; Kline JB et al., Eur J Immunol 48:1872-1882, 2018; Grasso L et al., Oncol Lett 23:2, 2022). In addition, these tumor-produced factors have also been shown to bind to the antibody component in antibody-drug conjugates (ADCs), thereby 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 co-regulated by the binding of antibodies to cell surface antigens, localizing the antibodies to epitopes on the cell surface. Once bound, the antibodies can bind to the Fc-activating receptors FCGR3A (CD16a) and FCGR2A (CD32a) on natural killer (NK) or dendritic / myeloid / monocytic cells, respectively (any cell involved in ADCC is referred to herein as an "immune effector cell"), to initiate ADCC or antibody-dependent cell phagocytosis (ADCP), and bind to the C1q complement initiation protein, causing the death of antibody-bound target cells through the classical complement CDC pathway (Reuschenbach M et al., Cancer Immunol Immunother 58:1535-1544, 2009). These effects have been observed in a variety of therapeutic antibodies, such as but not limited to rituximab, trastuzumab, cetuximab, pertuzumab, daratumumab, and alemtuzumab (Zhou X et al., The Oncologist 13:954-966, 2008; Hsu YF et al., Mol Cancer 9:1-8, 2010; Spiridon CI et al., Clin Cancer Res 8:1720-1730, 2002; Luo C et al., Sci Rep:46347, 2017; Casneuf T et al., Blood Adv 1:2105-2114, 2017).

[0005] Multiple reports have found that soluble and membrane-bound forms of ICAM-1 produced by tumors are associated with poor prognosis in patients with gastric cancer, non-small cell lung cancer, melanoma, breast cancer, colorectal cancer, multiple myeloma, and lymphoma (Maruo Y et al., Int J Cancer 100:486-490, 2002; Wu M et al., Pathol Res Pract 10.1016 / j.prp.2020.153029; Roland CL et al., Surgery 141:705–707, 2007). Some of these cancer indications use antibody-based therapies to kill tumors through immune effector mechanisms.

[0006] In this field, there is a continuing need to develop tools and agents to overcome humoral immune suppression and / or reduced ADC efficacy mediated by soluble and membrane-bound forms of ICAM-1 / CD54 (hereinafter referred to as ICAM-1), which applies not only to cancer patients but also to other ICAM-1-related humoral immune suppression diseases. Summary of the Invention

[0007] One aspect of the invention is an immunosuppression-tolerant human IgG1 antibody having one to four amino acid substitutions, as opposed to an immunosuppression-susceptible human IgG1 antibody without these amino acid substitutions. The immunosuppression of immunosuppression-susceptible human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0008] Another aspect of the present invention is a polynucleotide encoding an immunosuppressive tolerance human IgG1 antibody. The immunosuppressive tolerance human IgG1 antibody has one to four amino acid substitutions relative to an immunosuppressive susceptibility human IgG1 antibody without these amino acid substitutions. The immunosuppression of the immunosuppressive susceptibility human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0009] Another aspect of the present invention is a nucleic acid vector encoding an immunosuppressive tolerance human IgG1 antibody. The immunosuppressive tolerance human IgG1 antibody has one to four amino acid substitutions relative to an immunosuppressive susceptibility human IgG1 antibody without these amino acid substitutions. The immunosuppression of the immunosuppressive susceptibility human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0010] Another aspect of the present invention is a stable cell line comprising a nucleic acid vector. The stable cell line expresses an immunosuppressive tolerance human IgG1 antibody. The immunosuppressive tolerance human IgG1 antibody has one to four amino acid substitutions relative to an immunosuppressive susceptibility human IgG1 antibody without these amino acid substitutions. The immunosuppression of the immunosuppressive susceptibility human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0011] Another aspect of the present invention is a method for treating a patient suffering from a specific disease, wherein the level of ICAM-1 / CD54 expressed by the patient is elevated compared to that of a healthy population. The immunosuppressive tolerance human IgG1 antibody is administered to the patient in a standard form or as an antibody-drug conjugate (ADC). The immunosuppressive tolerance human IgG1 antibody has one to four amino acid substitutions relative to an immunosuppressive susceptibility human IgG1 antibody without these amino acid substitutions. The immunosuppression of the immunosuppressive susceptibility human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

[0012] Another aspect of the present invention is a method for treating a cancer patient or a patient with an inflammatory disease. A full-length human IgG1 antibody is administered to the cancer patient or the patient with an inflammatory disease. The antibody comprises a heavy chain having one to four amino acid substitutions in a motif in Kabat regions 367-425, mainly in Kabat regions 369-410, and these motifs are located within the heavy chain of the full-length human IgG1 antibody. In one embodiment, the amino acid substitution occurs at the amino acid residues YSKL (407-410) (SEQ ID NO:3).

[0013] Another aspect of the invention is a method for screening candidate antibodies to identify those that are resistant to ICAM-1 / CD54 immunosuppression. (a) A human cancer cell line expressing the antigen is contacted with a candidate IgG1 antibody that specifically binds to the antigen. The contact is carried out in the presence of ICAM-1 / CD54 (SEQ ID NO:1). (b) A human cancer cell line expressing the antigen is contacted with a candidate IgG1 antibody that specifically binds to the antigen. The contact is carried out 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 antibody in steps (a) and (b) is measured. (d) The antibody can be formulated into an antibody-drug conjugate (ADC). Target cell killing is determined by comparing the results achieved in step (a) and step (b).

[0014] Another aspect is a method for screening a candidate immunosuppression-tolerant human IgG1 antibody that has one to four amino acid substitutions relative to an immunosuppression-susceptible human IgG1 antibody to identify a candidate antibody that is tolerant to ICAM-1 / CD54 immunosuppression. The candidate immunosuppression-tolerant human IgG1 antibody is contacted with biotinylated human CD16a Fc receptor. The contact is carried out in the presence of sICAM-1 / CD54 as follows: SEQ ID NO:2 The immunosuppression-susceptible human IgG1 antibody is contacted with biotinylated human CD16a Fc receptor. The contact is carried out in the presence of sICAM-1 / CD54 as follows: SEQ ID NO:2 The binding of the candidate immunosuppression-tolerant and immunosuppression-susceptible IgG1 antibodies to the CD16a Fc receptor is determined.

[0015] Another aspect of the invention is a method for screening a candidate immunosuppression-tolerant human IgG1 antibody that has one to four amino acid substitutions relative to an immunosuppression-sensitive human IgG1 antibody to identify a candidate antibody that is tolerant to ICAM-1 / CD54 immunosuppression. The candidate immunosuppression-tolerant human IgG1 antibody is contacted with biotinylated human C1q protein. The contact is carried out in the presence of sICAM-1 / CD54 as follows: SEQ ID NO:2 The immunosuppression-sensitive human IgG1 antibody is contacted with biotinylated human C1q protein. The contact is carried out in the presence of sICAM-1 / CD54 as follows: SEQ ID NO:2 The binding of the candidate immunosuppression-tolerant human IgG1 antibody and the immunosuppression-sensitive human IgG1 antibody to the biotinylated human C1q protein receptor is determined.

[0016] Another aspect of the invention is a method for screening candidate immunosuppressive tolerance human IgG1 antibodies in the format of antibody-drug conjugates (ADCs), which have one to four amino acid substitutions relative to immunosuppressive sensitive human IgG1 antibodies, and can identify candidate antibodies that are tolerant to ICAM-1 / CD54 immunosuppression. The candidate immunosuppressive tolerance ADCs are cultured with homologous ICAM-1 expressing cells and non-ICAM-1 expressing cells, and the comparable killing of each cell type is determined.

[0017] Another aspect of the invention is a method for identifying tumor patients suitable for treatment with antibody-drug conjugates. The ICAM-1 expression of each tumor in a plurality of patients is detected. The detection determines whether each tumor in the plurality of patients expresses ICAM-1. If the tumor does not express ICAM-1, the patient is recommended to be treated with an IgG1 antibody-drug conjugate. The IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression. If the tumor expresses ICAM-1, the patient is recommended not to be treated with an IgG1 antibody-drug conjugate. Similarly, the IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression. At least one patient who does not express ICAM-1 can be selected for treatment with an IgG1 antibody-drug conjugate. The IgG1 antibody of the antibody-drug conjugate is sensitive to immunosuppression.

[0018] These and other aspects of the present invention will be apparent to those skilled in the art upon reading the specification, and they provide methods and formulation processes for improving antibody-mediated humoral immune responses and the efficacy of antibody-drug conjugates in ICAM-1 immunosuppressive diseases, including cancer and non-tumor diseases, to the art. Brief Description of the Drawings

[0019] Figures 1A and 1B. Identification of the binding of ICAM-1 to human IgG1. Briefly, 96-well ELISA plates were coated with (or without) 1-10 μg / mL human soluble ICAM-1 (sICAM-1) or human serum albumin (HSA) and incubated with 1-5 μg / mL rituximab, pertuzumab, or trastuzumab as previously described (Kline JB et al., OncoTarget 8:52045-52060, 2017). Each antibody was biotinylated and its binding to immobilized sICAM-1 was tested by ELISA. Using EZ-Link according to the manufacturer's instructions TMThe antibody was biotinylated with Sulfo-NHS-Biotin (Thermo Scientific). The biotinylated antibody was quantitatively read by Nanodrop and the 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 sICAM-1, similar to trastuzumab (P = 0.00009). To test the binding of sICAM-1 to immobilized IgG1, 5 μg / mL biotin-labeled sICAM-1 (Sino Biologicals) or biotin-labeled HAS generated with EZ-Link TM was incubated through a 96-well ELISA plate. As shown below, in Figure 1B, sICAM-1 could significantly bind to all antibodies similarly to pertuzumab (P = 0.001), but not to HSA. The experiments were performed with at least three replicate wells. Statistical analysis was performed using Student's t-test.

[0020] Figure 2A-2B. sICAM-1 inhibits CD16a activation. To test the ability of sICAM-1 to inhibit CD16a Fc--receptor activation and downstream ADCC, a Jurakt-CD16a-luciferase reporter system was used. As shown below, in Figure 2A, CD20-positive Daudi cells were used as target cells and CD16a activation was measured in the presence of increasing concentrations of sICAM-1 (0.1–10 μg / mL) using the anti-CD20 rituximab antibody according to the manufacturer's protocol. As shown, cultures treated with sICAM-1 showed inhibition of CD16a activation in a dose-responsive manner, with the highest dose having the strongest inhibitory effect (P = 0.0027). The experiments were performed with at least three replicate wells. Statistical analysis was performed using Student's t-test. In a similar manner, the ability of a panel of other antibodies to inhibit ADCC by sICAM-1 was analyzed. As shown below, in Figure 2B, 10 μg / mL sICAM-1 was able to inhibit CD16a activation of all antibodies at a dose of 1 μg / mL.

[0021] Figures 3A and 3B. Inhibition of CD16a Fc receptor and C1q binding. Inhibiting CD16a activation generally results in reduced binding of the CD16a Fc-receptor to IgG1 antibodies that bind antigen. To determine whether ICAM-1 binding to IgG1 would reduce CD16a Fc-receptor binding and complement-mediated binding of the C1q protein, an ELISA-based assay was performed to monitor their binding in the presence of sICAM-1. Briefly, pertuzumab was coated on 96-well plates and incubated 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). The experiment was presented with at least three replicate wells. Statistical analysis used the Student's T test.

[0022] Figures 4A - 4B. Domain mapping of ICAM-1 IgG1 binding. To localize the region of ICAM-1 that might bind to IgG1, human IgG1 fragments, F(ab’)2 and Fc fragments, were generated by papain digestion. Intact IgG1 and its fragments were immobilized on 96-well microtiter plates (2.5 μg / mL) and incubated with biotinylated sICAM-1 (0.5 μg / mL). As shown in Figure 4A, sICAM-1 specifically bound to the Fc domain (P = 0.00081, compared to HSA). The experiment was presented with at least three replicate wells. Statistical analysis used the Student's T test. Additionally, 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, while the other fragments did not show binding. This further localized the ICAM-1 binding domain to the CH2-CH3 region.

[0023] Figures 5A - 5C. IgG1 has heavy chain deletions and mutagenesis. To further define the region of ICAM-1 binding to IgG, GST fusion proteins consisting of the Fc domain (hinge region to C-terminus, including a C-terminal FlagTag) were generated and used for competitive ICAM-1-IgG1 binding by ELISA. The deletion numbering is based on the Kabat residue positions within the IgG1 heavy chain as follows. In Figure 5A, constructs numbered 20 - 24 were also expressed as full-length variants of rituximab. As shown in Figure 5A, the GST deletion mutants from Kabat 411 to the C-terminus still bound significantly to sICAM-1 (P = 0.000144, construct numbers 2 and 3). In contrast, mutants from Kabat 391 to 401 (construct numbers 4 and 5) lost their ICAM-1 binding ability. Figure 5B shows the representative experimental results using proteins of construct numbers 3 - 5. To further delimit this region, multiple alanine and / or glycine substitution mutants were generated, both as GST fusion proteins and as full-length rituximab antibodies, for competitive binding of ICAM-1 to the wild-type IgG1 heavy chain (Figure 5A, construct numbers 6 - 24). As shown in Figure 5C, the mutant heavy chains containing alanine and / or glycine substitutions (Kabat substitutions 407 - 410) completely lost their ability to compete with wild-type IgG1 (construct numbers 22 and 24), thus identifying the Kabat 407 - 410 region as the essential region for ICAM-1 binding. The test results presented are the values of three repeated measurements, and statistical data were determined using the Student's T-test.

[0024] Figure 6. A schematic diagram of the IgG1 ICAM-1 binding region is shown in the context of the CH3 domain and other functional binding sites for CD16a Fc-receptor, C1q, and FcRN binding.

[0025] Figure 7. Bioassay analysis of saponin-conjugated trastuzumab and pertuzumab (hereinafter referred to as ZAP) antibodies killing HER2 + Bioassay analysis of HCT116 wild-type (HCT116-WT) and HCT116 ICAM-1 knockdown (HCT116-ICAM1-KO) cells. As shown, compared with HCT116-WT cells, both trastuzumab-ZAP and pertuzumab-ZAP could significantly kill HCT116-ICAM1-KO cells (P < 0.025), indicating that cell membrane-expressed ICAM-1 has a negative impact on the internalization and target cell toxicity of antibody-toxin conjugates such as ADCs. The test results presented are the values of three repeated measurements, and statistical data were determined using the Student's T-test.

[0026] Figure 8. ELISA analysis of rituximab within the sICAM-1 binding domain with heavy chain CH3 modification. A competitive ELISA assay was performed to test the binding of sICAM-1 to parental rituximab (RTX) and RTX with residue modifications at positions 369-372, 374-377, and 407-410 (referred to as RTX-FARV). As shown, modifying the amino acids within these three regions resulted in a significant decrease in the binding of sICAM-1 to rituximab (P << 0.00065). The detection results represent the values of three repeated measurements, and statistical data were determined using the Student's T-test.

[0027] Figure 9. Biometric analysis of parental rituximab (RTX) and RTX-FARV in the presence of sICAM-1. In the presence or absence of 10 μg / mL sICAM-1, parental rituximab (RTX) or modified RTX (RTX-FARV) was used to test the activation of Jurkat-CD16a reporter cells against CD20 + Daudi target cells. As shown, both antibodies activated Jurkat-CD16a signaling, and the addition of sICAM-1 significantly inhibited the activation of RTX against Jurkat- CD16a compared to RTX-FARV (P = 0.0015). The detection results represent the values of three repeated measurements, and statistical data were determined using the Student's T-test. Reference electronic sequence listing

[0028] The content of the electronic sequence listing (sequencelisting.xml; size: 16,000 bytes; creation date: August 15, 2022) is hereby incorporated by reference in its entirety into this document. Detailed description of the invention

[0029] To determine whether any antibody used in antibody therapies that kill tumors by immune effector mechanisms is negatively affected by ICAM-1, we tested the physical binding of ICAM-1 to these antibodies. We also tested the humoral immune effector activity against tumor cells under immunosuppressive conditions. We found that ICAM-1 can bind to IgG1-type antibodies and have a negative impact on their humoral immune effector activity. In addition, the binding of ICAM-1 can affect the potency of antibody-drug conjugates (ADCs). The binding of ICAM-1 to antigen-bound ADCs on the cell surface slows down the internalization rate of the ADC, thereby having a negative impact on ADC efficacy (see Liao, MZ et al. Clinical Pharmacology and Therapeutics 110:1216-1230, 2021).

[0030] We found that the ICAM-1 protein can bind to a specific region in the CH3 domain of human IgG1-type antibodies and inhibit their immune effector activity. We show here that modification of this region (the IC1-binding region) can render IgG1 antibodies resistant to ICAM-1 immunosuppression. Use of ICAM-1-binding modified antibodies in tumor patients with overexpression of ICAM-1 can enhance or promote their therapeutic effects (see Maruo Y et al., Int J Cancer 100:486-490, 2002; Wu M et al., Pathology Research and Practice 10.1016 / j.prp.2020.153029; Roland CL et al., Surgery 141:705–707, 2007). As an example of this approach, we have generated ICAM-1-binding modified rituximab (SEQ ID NO:4 and 5), trastuzumab (SEQ ID NO:6), pertuzumab (SEQ ID NO:7), cetuximab (SEQ ID NO:8), and daratumumab (SEQ ID NO:9). Modification of the ICAM-1 binding site can be used to engineer the Fc domain of any IgG1-type antibody to generate antibodies resistant to ICAM-1, thereby improving therapeutic efficacy.

[0031] We have demonstrated that modification of Kabat residues 367-425 within the CH3 domain, preferably modification of Kabat residues 369–410 (the IC1-binding domain), can render the affected antibodies resistant to ICAM-1 immunosuppression. For example, the modification can be within residues 369-372, 374-377, and / or 407-410 of an IgG1-type antibody. These ICAM1-tolerant (also known as IC1-modified) antibodies are able to overcome humoral immunosuppression caused by the ICAM-1 protein. They can be used to treat cancer and other ICAM-1 immunosuppressive diseases. While not wishing to be limited to any particular theory or mechanism of action, the applicants believe that the ICAM-1 protein binds to Kabat residues 369-410 of the IgG1-type antibody and residues surrounding them, and disrupts antibody-mediated humoral immune responses through immune effector cells and / or the complement system. These disruptions include inhibition of C1q-antibody (classical antibody-complement) complexes and / or binding of the antibody to Fc-activating receptors (CD16a, CD32a, CD64a) on immune effector cells (such as but not limited to natural killer cells, dendritic cells, monocytes, and myeloid cells). These disruptions lead to downstream inhibition of CDC, ADCC, and ADCP. Changes in other parts of the three-dimensional structure of the antibody molecule close to the IC1-binding domain may also affect ICAM-1 binding. Therefore, proximity to the IC1-binding domain can be evaluated not only based on the primary structure of the antibody, but also based on its secondary, tertiary, and quaternary structures.

[0032] In addition, recent studies have shown that cell surface proteins bound to antibodies may negatively affect the efficacy of antibody-drug conjugates (ADCs) due to reduced internalization (Nicolaides NC et al., PLoS ONE DOI.org / 10.1371 / journal.pone.0285161, 2023). The cell surface expression of ICAM-1 may affect ADCs in a similar manner. Therefore, using IC1-modified antibodies may also improve the ADC treatment effect for tumors expressing ICAM-1.

[0033] The methods described herein can be used to develop other anti-ICAM-1 antibodies that target tumor-associated antigens (such as but not limited to CD20, CD38, HER2, or EGFR) to overcome humoral immune suppression or ADC inhibition. IC1-modified antibodies capable of circumventing ICAM-1-mediated humoral immune responses and / or ADC inhibition can be used in preclinical studies, human testing, and clinical practice.

[0034] For therapeutic applications, IC1-modified antibodies can be administered alone or in combination with other standard therapeutic drugs. They can be administered before, during, or after the administration of IC1-modified antibodies or ADCs.

[0035] Combination preparations formed during the process of these methods may be useful diagnostically or therapeutically. The combination preparation can be a combination of proteins with amino acid alterations in domains affected by other tumor immunosuppressive proteins, such as CA125 / MUC1 on rituximab (SEQ ID NO:14) (Grasso L et al., Oncology Letters 23:2, 2022). Other examples include IC1 modifications to the Fc domain, which can alter the biological properties of ADCC, CDC, or FcRN, all of which are well known to those skilled in the art. Any of the antibody selections described herein can be used to generate diagnostic or therapeutic combination preparations. These antibodies can also be used analytically as laboratory reagents.

[0036] The method of developing a composition by modifying the IC1 binding domain described herein is applicable to any antibody whose dynamic structure is altered by ICAM-1 binding, thereby inhibiting the humoral immune response and / or causing antibody internalization.

[0037] The "dynamic structure" of an antibody or protein refers to the three-dimensional structure of the antibody at a given point in time, where that point in time coincides with the binding of the antibody to another protein or agent, and the antibody structure before that point in time changes to a different structure after the antibody binds to another protein or agent.

[0038] Antibody-based methods continue to be used for the treatment of various cancers, as well as inflammatory and infectious diseases, while tumors utilize various pathways to evade the host's immune defenses. Therefore, it is important to identify agents and regions within the affected antibodies that can be modified to overcome humoral immune suppression and / or the effects of affected antibody internalization. These agents or combination formulations and methods of modified antibodies can select lead antibodies that may overcome ICAM-1-induced immunosuppression and / or reduced internalization, and contribute to patient screening. For example, patients can be screened to determine if they have high levels of ICAM-1 expression. For those patients who do have high levels of ICAM-1 expression, IC1-modified antibodies against tumor-specific antigens (i.e., rituximab (CD20 antigen); trastuzumab and pertuzumab (HER2 antigen); cetuximab (EGFR antigen); daratumumab (CD38 antigen), etc.) can be administered to overcome the inhibitory effects of ICAM-1 on the humoral response and / or internalization, which may affect ADC-formatted antibodies.

[0039] In the presence of ICAM-1, the dynamic structures of IgG1-type antibodies can be screened, and ICAM-1 can affect their dynamic structures and inhibit their downstream immune effector functions and / or internalization when binding to its cell surface target antigen. Figures 1A-1B provide examples of screening for ICAM-1 binding, and the following examples and figures illustrate how to modify such ICAM-1-sensitive antibodies to make them resistant to immunosuppression.

[0040] In one embodiment, human cancer cells expressing ICAM-1 can be used to detect the ICAM-1 immunosuppressive effects of wild-type versus IC1-modified antibodies.

[0041] In another embodiment, human cancer cells expressing ICAM-1 can be used to detect the antibody internalization rate and / or potency of wild-type versus IC1-modified antibody-drug conjugates (ADCs).

[0042] IC1-modified antibodies can contain one or more amino acid changes within or near the IC1-binding domain region (Kabat residues 369-410) of the human IgG1 heavy chain. Proximity can be primary, secondary, tertiary, or quaternary structure. The amino acid changes may be between Kabat residues 367-425. The ability of IC1-modified antibodies to overcome ICAM-1 humoral immune suppression of ADCC, ADCP, or CDC, as well as the inhibited ADC activity, can be tested.

[0043] In a method for measuring the effectiveness of IC1-modified antibodies to overcome ICAM-1 humoral immune suppression, the ability of the antibody to directly bind ICAM-1, and / or the ability of the antibody to bind CD16a or C1q protein in the presence of ICAM-1, is tested by ELISA or other methods known in the art.

[0044] In another method for measuring the ability of an IC1-modified antibody to effectively overcome the inhibition of an ADC-format antibody by ICAM-1, the direct ICAM-1 binding and / or the target cell internalization rate and / or the ADC target cell killing rate of the antibody are tested using methods known to those skilled in the art and compared with the IC1-modified format.

[0045] Functional methods can be used to measure the effect of an IC1-modified antibody in overcoming ICAM-1-mediated humoral immune suppression via ADCC, ADCP, or CDC. ICAM-1 can be produced by target cells or added as an exogenous "factor" in soluble form. The term "effect" generally refers to a 10% or greater change in the ADCC, ADCP, or CDC target cell killing rate when the agent is cultured with the parental antibody and the IC1-modified antibody. Depending on the antibody, this change may refer to at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0046] In this specification and the claims of this document, various terms and phrases related to the aspects described in the accompanying description are used. Unless otherwise clearly stated, these terms should be interpreted in their ordinary meaning in the art. Other clearly defined terms should be interpreted in a manner consistent with the provided definitions.

[0047] In this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms of "a", "an", and "the" also include plural referents. For example, reference to "a cell" may include a combination of two or more cells, and so on. The "probe" mentioned may include the parental antibody or the IC1 antibody, or an independent antibody against an antigen to monitor the humoral immune response by any assay method known to those skilled in the art.

[0048] When referring to a quantified value (such as a quantity, a period of time, etc.), the term "about" is intended to cover variations of no more than ±9% relative to the specified value, such variations being suitable for practicing the disclosed method. Unless otherwise stated, all values representing the amounts of reagents used in the specification and claims (such as molecular weight, molar concentration, reaction conditions, percentages, etc.) should be understood to be quantified by the term "about" in all cases. Accordingly, unless otherwise stated, the numerical values in the following specification and the listed claims are approximate values and may vary depending on the desired characteristics of the combination formulation and / or the method sought in the present invention. At a minimum, each numerical value should be taken by the reported significant digits and using the ordinary rounding method known to those skilled in the art, and this is not intended to limit the scope of the present application.

[0049] The term "antibody" as used herein is used in a broad sense and encompasses immunoglobulins (also referred to as "Ig") or antibody molecules, such as polyclonal antibodies (also referred to as pAbs), monoclonal antibodies (also referred to as mAbs, including murine, human, humanized, and chimeric mAbs), bispecific antibodies (also referred to as BSPs), antibody fragments, and antibody-drug conjugates (also referred to as ADCs). Generally speaking, an antibody is a protein or polypeptide chain that binds to a specific antigen. An antigen is a structure that is specifically recognized by a specific antibody. A classical antibody consists of a heterotetramer of glycosylated proteins, composed of two light chains and two heavy chains linked by complex disulfide bonds and hydrogen bonds. The term "its disulfide bonds" refers to the disulfide bonds contained within the heavy chain hinge region, which are well known to those skilled in the art. Each heavy chain has a variable domain (variable region) (VH), followed by a plurality of constant domains called CH1, CH2, and CH3, which together constitute the Fc domain. Each light chain has a variable domain (VL) and a constant domain; the constant domain of the light chain aligns with the first constant domain of the heavy chain, while the VL of the light chain aligns with the variable domain of the heavy chain. Antibody light chains of any species are divided into two different types based on the amino acid sequence within their constant domains, namely the κ type and the λ type.

[0050] Immunoglobulins are classified into different classes or isotypes based on the type of their Fc domain, namely IgA, IgD, IgE, IgG, and IgM, and these classes depend on the sequences contained within the constant regions of their heavy chains. The IgA and IgG isotypes are further divided into subclasses, including isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4.

[0051] The immunoglobulin VL or VH region consists of "framework" (FW) regions interrupted by three regions that are complementary determining due to sequence variation (CDRs), and these CDRs are the "antigen-binding sites", as reported (Wu TT and Kabat EA. J. Exp. Med. 132:211-250, 1970). Generally speaking, the antigen-binding site consists of six CDRs, three of which are located in the variable region of the heavy chain (CDRH1, CDRH2, CDRH3), and three are located in the variable region of the light chain (CDRL1, CDRL2, CDRL3) (Kabat EA et al., 5th 版 , PHS, National Institutes of Health, Bethesda, Md., 1991).

[0052] The "Kabat" numbering refers to a scheme for numbering amino acid residues in an antibody based on the variable region.

[0053] "Specifically binds" means that the binding affinity of an antibody or antigen-binding fragment to an antigen (including the sequence contained within the antibody itself) is greater than its binding to other antigens. In general, the equilibrium dissociation constant K of a specific antibody or antigen-binding fragment binding to a target antigenD about 5x10 -8 M or lower.

[0054] An "antigen" is a substance to which an antibody or antibody fragment binds specifically. This includes binding to a target antibody or target protein.

[0055] The term "antibody dynamic structure" refers to any structural change that may affect the humoral function of an antibody (i.e., Fc receptor or C1q binding, etc.).

[0056] The term "monoclonal antibody (mAb)" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or phage clone, and not to the method of its production. Thus, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology and may also include recombinant methods.

[0057] A "full-length antibody" refers to an IgG1-type antibody containing a complete variable region and heavy chain region, usually found in rodent or human serum or produced by recombinant DNA methods.

[0058] A "Fab domain" refers to any antibody sequence at the amino terminus of the antibody hinge disulfide region known to those skilled in the art.

[0059] An "Fc domain" refers to any antibody sequence at the carboxyl terminus of the antibody Fab domain, including the antibody hinge disulfide region known to those skilled in the art.

[0060] The "affected domain" or "ICAM-1 binding domain" or "IC1 region" refers to the amino acid sequence located in and around the human IgG1 Kabat 369-410 region, for example, the amino acid sequence located within the Kabat 407-410 (SEQ ID NO:3) region. The affected domain may be located within the CH3 domain, i.e., Kabat residues 367-425.

[0061] The term "parent antibody" refers to a human IgG1-type antibody composed of a wild-type IgG1 heavy chain sequence.

[0062] The term "IC1-modified antibody" or "IC1 antibody" refers to an antibody consisting of a heavy chain containing one or more amino acid changes located within or near the ICAM-1 binding domain. Substitutions within Kabat region 367-425 (CH3 domain) or Kabat region 369-410 (including region 407-410) can be made with 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 residues within the Kabat 367-425 region can be substituted.

[0063] The term "ICAM-1 tolerance" refers to an IC1-modified antibody that is superior to the parental antibody in terms of internalization in the form of ADCC, ADCP, CDC, and / or ADC.

[0064] The term "agent" refers to any protein or chemical substance capable of blocking or reducing the immune effector function of an antibody.

[0065] The term "affected antibody" refers to an antibody whose humoral immune function or internalization / antibody-drug conjugate target cell killing effect is reduced by ICAM-1.

[0066] The term "rituximab" refers to the antibody approved by the FDA [FDA reference number: 4274293].

[0067] The term "cetuximab" refers to the antibody approved by the FDA [FDA reference number: 4422941].

[0068] The term "trastuzumab" refers to the antibody approved by the FDA [FDA reference number: 4090445].

[0069] The term "pertuzumab" refers to the antibody approved by the FDA [FDA reference number: 3384285].

[0070] The term "daratumumab" refers to the antibody approved by the FDA [FDA reference number: 4924146].

[0071] The term "CD20" refers to a human cell surface protein expressed by B cells and is the target antigen specifically bound by rituximab.

[0072] 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.

[0073] The term "EGFR" refers to a human cell surface protein expressed by epithelial cells and is the target antigen to which cetuximab specifically binds.

[0074] The term "CD38" refers to a human cell surface protein expressed by lymphocytes and is the target antigen to which daratumumab specifically binds.

[0075] The term "CA125" refers to the gene product produced by the MUC16 gene (HGNC: 15582; OMIM: 606154) and exists in soluble and membrane-bound forms. It binds to antibodies and affects the humoral immune function of the binding antibodies (Kline JB et al., Oncotarget 8:52045-52060, 2017).

[0076] The term "ICAM-1" refers to the gene product produced by the ICAM-1 / CD54 gene (HGNC: 5344; NCBI reference sequence: NG_012083.1) and exists in soluble and membrane-bound forms. It binds to CD11a / CD18 and CD11b / CD18.

[0077] The terms "cancer", "malignant", "disorder" and "tumor" are well known in the art and refer to the presence of cells with uncontrolled cell growth and morphological characteristics different from normal cells of similar origin. Malignant refers to cancer cells that can cause morbidity and / or death. As used herein, "cancer" and "tumor" include pre-cancerous and malignant types.

[0078] As used herein, the term "soluble" refers to a protein or non-protein agent that is not attached to the cell cell membrane. For example, a soluble agent can be shed, secreted or exported from normal or cancer cells into biological fluids, including serum, whole blood, plasma, urine or cell microfluids, and also includes tumor cells.

[0079] The "level" of a specified protein agent used (including IC1-modified antibodies and ICAM-1) refers to the amount determined by measuring protein levels in vitro or in vivo using any method known in the art. Such methods include gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high performance diffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double diffusion), solution-phase assays, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Förster resonance energy transfer, electrochemiluminescence immunoassay, etc. In the examples, probe-based techniques are used to determine the level of ICAM-1.

[0080] The term "humoral immune suppression" refers to any antibody, antibody fragment, bispecific antibody (BSP), or antibody-drug conjugate (ADC) that directly binds to ICAM-1 and whose dynamic structure is altered. ICAM-1 can be in membrane-bound form or soluble form.

[0081] The term "bispecific antibody (BSP)" refers to any antibody that can bind two or more different antigens. BSP can include at least, but not limited to, two full-length antibodies, one full-length antibody and one single-chain antibody, or two single-chain antibodies, where each antibody binds to a different antigen or a different epitope on the same antigen.

[0082] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to an in vitro or in vivo process in which an antibody can bind to an antigen on the cell surface and then bind to immune effector cells through sequences within the Fc domain of the antibody, leading to the release of toxins by the immune effector cells, thereby killing the cells bound by the antibody.

[0083] The term "complement-dependent cytotoxicity (CDC)" refers to an in vitro or in vivo process in which an antibody can bind to an antigen on the surface of eukaryotic or prokaryotic cells and then bind to the C1q protein through sequences within the Fc domain of the antibody, thereby initiating the classical complement cascade and killing the cells bound by the antibody.

[0084] The term "opsonization" refers to a process called antibody-dependent cell phagocytosis (ADCP), in which an antibody can bind to an antigen on the cell surface and then contact immune cells through sequences within its Fc domain, leading to the phagocytosis, consumption, and ultimately killing of the cells bound by the antibody.

[0085] The term "native" or "naive" or "classical" antibody or "antibody form" refers to a full-length parental antibody or IC1-modified antibody that has not undergone any chemical modification.

[0086] The term "antibody-drug conjugate (ADC)" refers to an antibody conjugated to a chemical or biological agent either covalently or non-covalently. These include radionuclides, nucleic acids, immunotoxins, small molecule compounds. These conjugated agents can be cytotoxic, cytostatic or diagnostic (signals detectable by fluorescence, luminescence, radioimaging or enzyme signal emission).

[0087] The term "pharmacokinetics (PK)" refers to the time during which an antibody maintains a steady-state concentration after administration to a subject.

[0088] 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 the actions and effects of these drugs and their biochemical structures when administered to a subject.

[0089] The term "pharmacology (PL)" refers to the known actions of an antibody to control or kill diseased cells in vitro or in vivo.

[0090] The term "sample" refers to a group of similar fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues present in a subject. Fluids may include biological fluid solutions in contact with a subject or biological source, including cell and organoid culture media, urine, saliva, lavage fluids, etc.

[0091] The term "control sample" or "control protein" refers to any clinically or non-clinically relevant control sample, including for example samples from healthy subjects not suffering from a specific cancer type or cells different from their parental cells.

[0092] The term "control level" refers to an accepted or predetermined level (quantity) used to compare with the level of the same substance agent in a sample from a subject or the level used in an in vitro assay.

[0093] As used, the term "difference" refers to the difference between the signal of an antibody under control conditions and its signal when binding to ICAM-1, typically any difference that can be determined using commonly used statistical methods in the art and is at least 10% or greater relative to the control group. 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%.

[0094] The terms "inhibit", "inhibition", "suppress" or "reduce" refer to reducing to a statistically measurable amount or completely preventing.

[0095] In the methods described, the term "functional" in the context of an antibody or antibody-containing moiety (such as an ADC, BSP, etc.) indicates that the IC1-modified antibody reduces the binding of ICAM-1 to an IgG1-type antibody, respectively, and / or is more capable of killing target cells in vitro or in vivo than the parental antibody alone.

[0096] The term "target cell" refers to a eukaryotic or prokaryotic cell or cell population that expresses the antigen of a specific antibody or antibody-containing moiety.

[0097] The term "pharmaceutically acceptable" refers to a substance that can be administered to a patient from a pharmacological and toxicological perspective, and is manufactured using methods known to those skilled in the art. These include drugs for animals and humans that are approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopeias. The term "pharmaceutically compatible components" refers to pharmaceutically acceptable diluents, adjuvants, excipients, or matrix carriers administered together with an anticancer agent. A "pharmaceutically acceptable carrier" is a matrix that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host.

[0098] The terms "effective amount" and "therapeutically effective amount" are used interchangeably and are used when the drug dose is sufficient to produce an enhanced clinical effect in a patient. The effective amount of the agent is administered according to the method in the "effective regimen" described herein. The term "effective regimen" refers to a combination of drug dose and administration frequency sufficient to achieve enhanced clinical efficacy in a patient suffering from a specific cancer. Enhanced efficacy refers to the improved clinical outcome obtained when a patient receives a drug that is more capable of overcoming the disease or improving the clinical efficacy of the effective regimen than the base compound. As described in the context herein, the effective amount refers to the dose of the IC1-modified antibody required to show efficacy or a difference compared to the parental antibody.

[0099] The terms "patient" and "subject" are used interchangeably and refer to humans and other non-human animals, including veterinary subjects, that are treated with a therapeutic agent. The term "non-human animal" includes all vertebrates. In the examples, the subject is a human.

[0100] A "therapeutic agent" generally contains substantially no undesired contaminants. This means that the agent is typically at least about 50% w / w (weight / weight) pure and is substantially free of interfering proteins and contaminants.

[0101] The term "immune effector cell" refers to any cell, including but not limited to natural killer (NK) cells, myeloid cells, monocytes, and / or dendritic cells, which can confer antibody-dependent cell cytotoxicity (ADCC) or phagocytosis (ADCP, opsonization) upon contact with an antibody-bound target cell. The cells can be purified or present in a mixture in the form of peripheral blood mononuclear cells (PBMC).

[0102] Inflammatory diseases include autoimmune diseases, rheumatoid arthritis, granulomatosis with polyangiitis, idiopathic thrombocytopenic purpura, pemphigus vulgaris, myasthenia gravis, atherosclerosis, and Epstein-Barr virus-positive mucocutaneous ulcers.

[0103] Infectious diseases include viral, bacterial, protozoal, and parasitic diseases. Viral diseases include, but are not limited to, influenza, AIDS, meningitis, pneumonia, herpes, human papillomavirus, respiratory syncytial virus, parainfluenza virus, Ebola virus, measles, chickenpox, and shingles. Bacterial diseases include, but are not limited to, tuberculosis, whooping cough, legionellosis, pneumonia, and urinary tract infections. Protozoal diseases include malaria, giardiasis, Chagas disease, amebiasis, trichomoniasis, trypanosomiasis, and toxoplasmosis. Parasitic diseases include, but are not limited to, filariasis, pediculosis, fascioliasis, schistosomiasis, and cryptosporidiosis.

[0104] The term "dysregulated cell" refers to any cell that is considered abnormal relative to a parental cell. These include transformed cells, malignant cells, virus-infected cells, and cells that grow autonomously through autoregulation.

[0105] The term "test antibody" refers to an antibody used to analyze immune effector activity.

[0106] The term "humoral response" refers to the antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity of a test antibody.

[0107] The term "screening" may refer to testing a protein capable of binding to ICAM-1 in the presence of an affected antibody or antibody-containing moiety (i.e., BSP, ADC, single-chain antibody, antibody fragment, etc.) and observing an enhanced biological response, monitoring the total cell number, or the killing mediated by ADCC, ADCP, or CDC. The term can also be used in other contexts to refer to detecting a large number of test elements to determine which of them have a specific property. Similarly, it can be used to refer to detecting patient samples with a specific property, such as elevated ICAM-1 RNA or protein.

[0108] The term "significantly(ly)" refers to a statistical result where the P-value determined by any procedure, including the student T-test, is less than 0.05. Composition of improved therapeutic antibodies and methods for developing and overcoming ICAM-1 / CD54-mediated humoral immune suppression and reduced efficacy of antibody-drug conjugates.

[0109] Binding of ICAM-1 / CD54 to human IgG1 antibodies results in reduced immune effector function. Engineered IgG1 antibodies can overcome the inhibition of humoral immune responses by ICAM-1 / CD54 and the inhibition of the efficacy of antibody-drug conjugates (ADCs) by the affected parental IgG1 antibodies. In some embodiments, methods for identifying ICAM-1 / CD54 non-reactive antibodies include generating IgG1 antibodies containing one or more amino acid changes within or around the affected ICAM-1 / CD54 binding domain (IC1 region) and testing whether the IC1-modified IgG1 antibodies have significantly improved biological activity when used to mediate ADCC, ADCP, or CDC killing or enhance ADC target cell killing of antigen-specifically expressing target cells. In certain embodiments, the IgG1 antibody contains one amino acid alteration. In other embodiments, the IgG1 antibody consists of two or more amino acid changes. In the presence of antibodies with mutations in amino acids within or near the IC1 region, functional ADCC, ADCP, CDC, ADC killing assays can be used to determine the optimal amino acid changes. These assays can employ primary cells or reporter cells, such as the Jurkat-CD16a ADCC reporter cell line. See Example 2 for examples. As shown in Figures 2 and 8, molecular detection methods can also be used to monitor the binding of CD16a Fc receptor or C1q protein to the antibody to identify ICAM-1-resistant IgG1 antibodies that can significantly overcome ICAM-1 humoral immune inhibition. These detection methods are commonly used by those skilled in the art, as shown in Figure 3.

[0110] IC1-modified antibodies may have similar or higher binding affinities compared to the parental IgG1 antibodies.

[0111] In some methods for identifying ICAM-1 / CD54-tolerant IgG1 antibodies, the antibody is added to a culture of target cells that naturally or recombinantly express IgG1-specific antigen and ICAM-1. Standard CD16a activation, ADCC, ADCP, or CDC killing assays can be used to monitor the culture to compare the humoral responses in the presence of IC1-modified antibody and parental antibody. A change of at least 10% is generally considered to have a significant effect on ADCC and / or CDC function. Depending on the antibody and assay used, a significant effect can also be defined as having at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75% change. The target cell line can be engineered to overexpress ICAM-1 by transduction with a human ICAM-1 expression construct or to reduce ICAM-1 expression by constructing an ICAM-1 shRNA knockout line with shRNA-specific constructs (SEQ ID NO: 10-13).

[0112] Cancer patients can be treated with IC1-modified IgG1 antibodies. For example, a patient may have a cancer that expresses CD20, such as Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, large cell lymphoma, or chronic lymphocytic leukemia. Multiple reports have found that elevated ICAM-1 expression in these cancer patients is associated with poor prognosis. Rituximab is one of the standard treatments for these cancers. In this case, the IC1-modified rituximab antibody may be an ideal therapeutic agent. The IC1-modified antibody may be formulated as an antibody-drug conjugate or remain in its native state.

[0113] It has been reported that overexpression of ICAM-1 in breast cancer and gastric cancer leads to a poor prognosis. The anti-HER2 trastuzumab and pertuzumab IgG1 antibodies have both been approved for the treatment of these types of cancers. In certain embodiments, IC1-modified trastuzumab or pertuzumab may be an ideal choice for treating patients with breast cancer or gastric cancer with overexpressed ICAM-1. The IC1-modified antibody can be formulated as an antibody-drug conjugate or exist in its native state.

[0114] An IC1-modified cetuximab that targets the EGFR protein can be used for treatment. Cetuximab has been approved for the treatment of colorectal cancer and head and neck cancer. Patients with these cancers who exhibit elevated ICAM-1 can benefit from IC1-modified cetuximab, making IC1-modified cetuximab an ideal choice. The IC1-modified antibody can be formulated as an antibody-drug conjugate or exist in its native state.

[0115] An IC1-modified daratumumab that targets the CD38 protein can be used for treatment. Daratumumab has been approved for the treatment of multiple myeloma. Patients with these cancers who exhibit elevated ICAM-1 can benefit from IC1-modified daratumumab, making IC1-modified daratumumab an ideal drug. The IC1-modified antibody can be formulated as an antibody-drug conjugate or exist in its native state.

[0116] Cancer patients who are positive for CD20, CD38, HER2 or EGFR and overexpress ICAM-1 / CD54 can be treated with IC1-modified rituximab, daratumumab, trastuzumab / pertuzumab or cetuximab antibodies respectively, which can be used in their native form or ADC form alone, or in combination with standard of care therapies. In some embodiments of the methods for treating patients with ICAM-1 overexpressing cancers described herein, an IC1-modified antibody is administered to patients with baseline ICAM-1 / CD54 levels above the normal range. In some embodiments of the methods for treating patients with ICAM-1 overexpressing cancers described herein, the method involves administering the IC1-modified antibody alone. In another embodiment, the IC1-modified antibody is co-administered with chemotherapy. The chemotherapy can be any chemotherapeutic agent that is considered standard treatment for a particular cancer at the time the patient is being treated. In the treatment methods described herein, the expression level of ICAM-1 / CD54 can be determined by any method known in the art and is defined by those skilled in the art as being within or above the normal range.

[0117] The IC1-modified antibody may have a change in one amino acid in the affected region (Kabat region 369-410). The IC1-modified antibody may have two or more amino acid changes in the affected domain. The IC1-modified antibody can be used in tumor patients who overexpress ICAM-1 / CD54 and express the antibody-targeted antigen. Cancers known to overexpress ICAM-1 / CD54 include Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, large cell lymphoma and chronic lymphocytic leukemia. Other cancers may also be suitable for this treatment, including but not limited to multiple myeloma, melanoma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer and head and neck cancer.

[0118] This method can be used in combination with other treatment modalities, such as surgery (e.g., debulking surgery), radiotherapy, targeted therapy, chemotherapy, immunotherapy, the use of growth factor inhibitors or anti-angiogenic factors. An IC1-modified antibody can be co-administered with ongoing surgery, chemotherapy or radiotherapy in the patient. Alternatively, the patient can undergo surgery, chemotherapy or radiotherapy before or after the administration of the IC1-modified antibody, with an interval of at least one hour to several months, such as at least one hour, five hours, 12 hours, one day, one week, one month or three months before or after standard of care treatment. In some cases, an effective therapeutic dose of a chemotherapeutic agent plus an IC1-modified antibody can be administered, such as rituximab, daratumumab, trastuzumab, pertuzumab, cetuximab, etc.

[0119] Before administering the IC1-modified antibody against the antigen expressed by the cancer, the subject may have undergone first-line surgical resection of the tumor or first-line chemotherapy for the cancer.

[0120] Therapeutic antibodies containing CDR sequences that direct the antibody to bind to the CD20 antigen and contain an IC1-modified heavy chain (e.g., SEQ ID NO: 4 or 5) can be used; antibodies against the HER2 antigen and containing an IC1-modified heavy chain (e.g., SEQ ID NO: 6 or 7); antibodies against the EGFR antigen and having an IC1-modified heavy chain (e.g., SEQ ID NO: 8); antibodies against the CD38 antigen and having an IC1-modified heavy chain (e.g., SEQ ID NO: 9), wherein each antibody contains one or more modifications within or near the ICAM-1 / CD54 binding domain (Kabat region 369–410). The antibody can be in the form of an ADC or the native form. Suitable antibodies can be administered to subjects with antigen-positive (relative to the therapeutic antibody) disease indications and ICAM-1 / CD54 levels above the normal range. Treatment can include surgery and standard treatments.

[0121] A variety of delivery systems can be used to administer the IC1-modified antibody, including intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes, as needed. The antibody can be administered by, for example, infusion or bolus injection and absorbed through epithelial or mucosal linings by systemic or local methods. Typically, the antibody is administered by intravenous infusion.

[0122] The IC1-modified antibody can be administered by injection using a syringe, catheter, or any implantable matrix or device.

[0123] The IC1-modified antibody can be combined with other drugs and administered in the form of a drug combination preparation, which contains a therapeutically or prophylactically effective amount of a therapeutic agent and one or more pharmaceutically acceptable or compatible components.

[0124] An effective therapeutic dose for treating or preventing cancer or non-oncological diseases can be determined by standard clinical techniques. In addition, in vitro assays can be selected to help determine the optimal dose range required for the IC1-modified antibody. The effective dose can be inferred from the dose-response curve of the IC1-modified antibody obtained from in vitro or animal model test systems.

[0125] For example, the toxicity and therapeutic effects of the IC1-modified antibody can be determined in cell culture or experimental animals by standard pharmacological procedures to determine the LD 50 (the dose lethal to 50% of the experimental animals) and the ED 50 (the dose having a therapeutic effect on 50% of the experimental animals) values. The dose ratio between toxicity and therapeutic effect is called the therapeutic index and can be expressed as LD 50 / ED 50The ratio. Agents with a larger therapeutic index are a suitable choice. When an agent exhibits toxic side effects, a delivery system can be used to deliver the agent to the targeted affected tissue site, minimizing potential damage to non-antigen-expressing cells and thus reducing side effects.

[0126] For example, the nucleic acid carrier can be a plasmid, virus, or subvirus. Ideally, the nucleic acid will be maintained in the desired host cell by having an effective origin of replication, but in some cases, transient expression may be required. Usually, an antibody modified with IC1 needs to be expressed, which requires an expression control sequence 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 all or part of the antibody are often formed. For example, the antibody fragment may be a complete light chain or heavy chain. The nucleic acid and vector can be used to create a cell line expressing the desired antibody. These cell lines can be used in the manufacturing process. Alternatively, cell lines expressing the antibody can be delivered so that they secrete the antibody directly to the patient. For example, these cell lines may be in situ in a subcutaneous device for protein production. The nucleic acid or vector can also be delivered to the subject in the form of gene therapy.

[0127] The dose and dosing schedule may vary depending on the concentration of the active drug, which may depend on the needs, body size, and condition of the subject.

[0128] According to one aspect of the present invention, a method for treating a cancer patient or a patient suffering from an inflammatory disease is provided. An ICAM-1 modified (referred to herein as IC1) human IgG1-type antibody is administered to a cancer patient or a patient suffering from an inflammatory or infectious disease. The administered protein is resistant to ICAM-1 binding and enhances the effect of the therapeutic antibody, whether or not it has an additional modified Fc domain. The IC1-modified antibody may contain 1, 2, 3, or 4 amino acid modifications, or a combination of amino acids, which modify the sequence of the wild-type human IgG1 Fc domain within the IC1 binding domain (Kabat residues 369-410), or delete part of the IC1 binding domain. The antibody may have one or more amino acid changes within or outside the IC1 binding domain (Kabat region 369-410). Any antibody containing a modification in the 367-425 region, whether or not it includes a modification within Kabat region 407-410, is herein referred to as an IC1-engineered antibody or an IC1-modified antibody.

[0129] Another aspect of the present invention relates to modifying one or more amino acids in the human IgG1 CH3 domain (Figure 6); these modified amino acids reduce the binding of ICAM-1 but still retain the ability to bind to the CD16a Fc receptor and C1q protein.

[0130] It is possible to generate IC1-engineered IgG1 antibodies against tumor proteins. Tumor proteins may include, but are not limited to, BCMA, CD19, CD20, CD22, CD30, CTLA-4, CD38, epidermal growth factor, fibroblast growth factor 1, 2, 3, or 4, folate receptor α, HER2, mesothelin, PD-1, or PD-L1. All of these tumor proteins are known in the art.

[0131] It is possible to treat patients with cancer or inflammatory diseases with high expression of sICAM-1 or membrane-bound ICAM-1 compared to healthy individuals. The treatment regimen includes using an IC1-modified antibody that targets an antigen specific to the diseased cells. Typically, the drug can be administered daily, once a week, once every three weeks, or once a month.

[0132] Patients with non-Hodgkin lymphoma (NHL) can be treated with IC1-modified rituximab containing a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 4), or with rituximab refractory to CA125 (Grasso L et al., Oncology Letters 23:2, 2022) (SEQ ID NO: 5) heavy chain. The IC1-modified rituximab antibody can be used alone or in combination with other chemotherapeutic drugs. The antibody binds to CD20-positive target cells and triggers a humoral immune response, such as ADCC, ADCP, or CDC, to kill the bound cells. The antibody can be in the form of an ADC or a natural form.

[0133] Patients with HER2-positive breast cancer, gastric cancer, or head and neck cancer can be treated with IC1-modified trastuzumab containing a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO: 6). The IC1-modified trastuzumab antibody can be used alone or in combination with other chemotherapeutic drugs. The antibody binds to HER2-positive target cells and triggers a humoral immune response, such as ADCC, ADCP, or CDC, to kill the bound cells. The antibody can be in the form of an ADC or a natural form.

[0134] Patients with HER2-positive breast cancer, gastric cancer, or head and neck cancer can be treated with IC1-modified pertuzumab containing 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 drugs. The antibody binds to HER2-positive target cells and triggers a humoral immune response, such as ADCC, ADCP, or CDC, to kill the bound cells. The antibody can be in the form of an ADC or a natural form.

[0135] Patients with colorectal cancer or head and neck cancer can be treated with IC1-modified cetuximab comprising 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 drugs. The antibody binds to EGFR-positive target cells and elicits a humoral immune response, such as ADCC, ADCP, or CDC, to kill the bound cells. The antibody can be in the form of an ADC or a native form.

[0136] Patients with multiple myeloma can be treated with IC1-modified daratumumab comprising a wild-type light chain and an IC1-modified heavy chain (SEQ ID NO:9). The IC1-modified daratumumab antibody can be administered alone or in combination with other chemotherapeutic drugs. The antibody binds to CD38-positive target cells and elicits a humoral immune response, such as ADCC, ADCP, or CDC, to kill the bound cells. The antibody can be in the form of an ADC or a native form.

[0137] In the following examples, we show that ICAM-1 (SEQ ID NOS: 1 and 2) can directly bind to an immunoglobulin of the IgG1 type and inhibit its immune effector functions. In addition, we have identified a specific region on the human IgG1 Fc domain (termed the “IC1” binding region) that, when modified, renders the susceptible immunoglobulin tolerant to the immunosuppression by soluble ICAM-1 (sICAM-1) (SEQ ID NO:2). In addition, residues near the IC1 binding region can also be modified to generate anti-ICAM-1 antibodies.

[0138] The binding of IgG1 to membrane-bound ICAM-1 (mICAM-1) (SEQ ID NO:1) may be involved in inflammatory diseases. The wild-type IgG1 Fc domain or the IgG1 Fc domain can be IC1-modified and optionally further modified to enhance or reduce ADCC, ADCP, and / or CDC activities (Natsume A et al. Drug Design, Development and Therapy 3:7-16, 2009; Saunder KO. Frontiers in Immunology 10:1296-1316, 2019). In addition, the Fc region can be modified to enhance the binding to the neonatal Fc receptor (FcRN), which may improve its serum half-life (Dall’Acqua WF et al., Biochemistry 281:23514-23524, 2006). In addition, these modifications can be designed into various antibody forms, such as bispecific (BSP) antibodies and antibody-drug conjugates (ADCs), to improve their therapeutic activities. Example 1 - Screening for proteins that inhibit the humoral immune effector functions of antibodies

[0139] Recent research has found that a group of proteins produced by tumors inhibit antibody-mediated killing through 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 Lett 23:2, 2022). In addition, antibodies that bind to such proteins have been shown to be negatively affected in the form of antibody-drug conjugates (Nicolaides NC et al., PLoS ONE DOI.org / 10.1371 / journal.pone.0285161, 2023). This immunosuppressive activity appears to occur through direct binding to specific regions of the affected antibodies. To identify other humoral immunosuppressive proteins, molecular and biological assays have been used to test candidate proteins reported by other researchers that are produced by various types of tumors and are associated with poor prognosis to determine their potential as antibody immunosuppressants. Multiple research groups have reported that the ICAM-1 protein is associated with multiple cancers and poor prognosis. To evaluate its effect on antibody immune effector activity, antibody binding to soluble ICAM-1 (sICAM-1) was evaluated. The screening assay used 96-well plate ELISA, where the wells were coated with sICAM-1 protein (Sino Biologicals) (SEQ ID NO:2) and incubated with biotinylated human antibodies to measure binding. As shown below, in Figure 1A, the wells containing sICAM-1 were bound by all tested IgG1 antibodies, while the wells coated with human serum albumin (HSA) protein as a negative control did not show such binding. Then, ICAM-1 / IgG1 binding was tested using immobilized IgG1 or IgM class antibodies and biotinylated sICAM-1 or HSA. As shown below, in Figure 1B, the wells containing IgG1-type antibodies and incubated with sICAM-1 showed strong binding compared to the wells containing control proteins (IgM, HSA), demonstrating that sICAM-1 is capable of directly binding to IgG1-type antibodies.

[0140] The binding of a protein to an IgG1 antibody may or may not affect its ability to inhibit immune effector activity. To test the effect of the binding of sICAM-1 to an IgG1 antibody on immune effector activity, we used a bioassay to monitor ADCC activity. Reports have shown that membrane-bound ICAM-1 plays a role in enhancing the ADCC of natural killer (NK) cells by binding to LFA, and blocking this interaction reduces its ADCC activity on target cells (Cooley S et al., Exp Hematol 27:1533-1541, 1999; Sanchez-Martinez D et al., Theranostics 8:3856-3869, 2018). To avoid the potential complexity of the ICAM-1 / LFA pathway in interpreting the ADCC effect mediated by sICAM-1IgG1, we used the Jurkat-CD16a-Luciferase (Jurkat-CD16a) system (Promega) to monitor the activation of CD16a, which is a prerequisite for ADCC activity of effector cells. In this experiment, CD20-positive Daudi cells were used as target cells, and rituximab (SEQ ID NO:4) against CD20 was used as the targeting IgG1 antibody. Briefly, 2x10 4 target cells were seeded in a black opaque 96-well plate overnight in R1 assay buffer (RPMI + L-glutamine + 1% ultra-low immunoglobulin serum) (Gibco). The next day, 1x10 5 Jurkat-CD16a effector cells were added together with 2.5 μg / mL of rituximab and different concentrations of sICAM-1 (ranging from 0 to 10 (g / mL) was added to the wells containing R1 detection buffer, maintaining an effector cell to target cell ratio of 5:1, and the culture plate was incubated at 37 °C and 5% CO2 for 16 hours. After incubation, the microplate was equilibrated at room temperature for 30 minutes, and then the activation of Jurkat-CD16a was measured using the BIO-GLO luciferase reagent according to the manufacturer's protocol (Promega). The activation of CD16a was quantitatively determined using a Varioskan LUX plate reader (ThermoFisher). As shown below, in Figure 2A, sICAM-1 had a significant dose-response effect on inhibiting rituximab-induced CD16a activation against Daudi cells (P < 0.003). To confirm the ability of sICAM-1 to act on other IgG1 antibodies, similar Jurkat-CD16a activation assays were performed at a concentration of 1 μg / mL using trastuzumab, pertuzumab, cetuximab, and rituximab (as a repeat control) in the presence of 10 μg / mL sICAM-1. For trastuzumab and pertuzumab, the human SK-BR-3 breast cancer cell line expressing HER2 was used as the target cell. For cetuximab, the A431 epidermoid cancer cell line expressing EGFR was used as the target cell. As shown below, in Figure 2B, sICAM-1 was able to inhibit CD16a activation of all four antibodies (P < 0.008). Toxicity assays of sICAM-1 on effector and target cells found that the cells were well tolerated when co-incubated at a concentration of 10 μg / mL, ruling out any potential artifacts in the above assays.

[0141] To demonstrate the effect of ICAM-1 on inhibiting IgG1 immune effector activity, isogenic ICAM-1 knockdown cells were generated using the HCT116 cell line expressing ICAM-1. Briefly, these cells were seeded at 7.5 × 10 in a 6-well plate 5Cells were generated by incubating overnight in 3 mL of complete medium RPMI (RPMI-1640 supplemented with 7.5% fetal bovine serum and 1% L-glutamine) at 37 °C and 5% CO2. The next day, the 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 were used as negative controls. After transfection, cells were selected using puromycin selection included in the expression construct to achieve stable construct integration. After selection, the cultures were subjected to single cell cloning in 96-well plates, and then protein lysate samples were screened for the absence of ICAM-1 expression by Western blotting using a 1:1000 dilution of anti-hICAM-1 rabbit polyclonal antibody (Sino Biologicals) and the method described previously. The knockdown clones were amplified for storage and tested for reduced IgG1 immune effector activity to confirm the immunosuppressive effect of sICAM-1 compared to the membrane-bound ICAM-1 in the homologous parental cells.

[0142] In addition, the inhibitory effect of cell monitoring membrane-bound ICAM-1 on the activity of IgG1 antibody-drug conjugates (ADCs) was used, because the physical interaction between ICAM-1 and the IgG1 Fc domain may inhibit internalization. Previous studies on antibodies in the form of ADCs have shown that proteins interacting with antibodies (such as MUC16 / CA125) can interfere with the internalization of ADCs and reduce their target cell toxicity (Nicolaides NC et al. PLoS ONE May 17, 2023; 18(5):e0285161). To determine the possible effect of ICAM-1 on antibodies in the form of ADCs, we adopted the ZAP streptavidin-saporin antibody conjugation system according to the manufacturer's (Advanced Targeting Systems) protocol. ZAP conjugates saporin with biotinylated antibodies, and once the antibody-saporin complex is internalized into target cells expressing the antigen, saporin is released and becomes toxic by inhibiting ribosomes (Polito, L, et al. Toxins 5:1698-1722, 2013). Briefly, both trastuzumab and pertuzumab antibodies were biotinylated using the EZ-link biotinylation reagent according to the manufacturer's (Thermo Scientific) protocol. Then, 100 nM biotinylated antibody and 100 nM streptavidin-saporin were added to RPMI 7.5% FBS growth medium, and 100 μL was added to each well of HCT116 wild-type and HCT-116-ICAM-1 knockdown cells seeded at 5,000 cells / well in clear 96-well microplates. Wells containing only 100 nM ZAP and no ZAP antibody were used as negative controls. The plates were incubated at 37 °C and 5% CO2 for 5 days, and then the killing effects of trastuzumab-ZAP and pertuzumab-ZAP were analyzed using crystal violet staining, and quantitative readings were taken at a wavelength of 576 nm using a Varioskan plate reader. As shown below, in Figure 7, compared with HCT116 wild-type (HCT116-WT) cells, trastuzumab-ZAP and pertuzumab-ZAP had a significant killing effect on HCT116-ICAM-1 knockdown cells (HCT116-ICAM1-KO) (P < 0.025). The wild and knockout cell lines expressed similar numbers of HER2 receptors. All experiments were repeated three times, and statistical analysis was performed using the Student's T test. The following results can be obtained from these data: The binding of ICAM-1 to antibodies in the form of ADCs may disrupt their internalization activity and target cell toxicity. The generation of antibodies lacking or carrying an altered ICAM-1-binding (IC1) domain is a useful invention for obtaining optimized ADCs. At the same time, it is also possible to screen whether the tumors of patients have ICAM-1 membrane expression, and tumors lacking ICAM-1 expression may be more suitable for ADC-based treatment.

[0143] The effect of tumor immunosuppressive proteins on antibody immune effector activity has previously been shown to be due to reduced binding of CD16a and / or C1q 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 evaluate the ability of sICAM-1 to inhibit the binding of these two 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 overnight with 1 μg / mL of pertuzumab or HSA (used as a negative control) in 0.05 M carbonate buffer at 4°C. The next day, the wells were washed with phosphate buffer (PB) at pH 7.2 and blocked with 5% BSA in PB for 1 hour, then washed three times in PB. Next, the wells were probed with 2.5 μg / mL of biotinylated soluble human CD16a (Sino Biologicals) or 1 μg / mL of biotinylated C1q (Sigma), and then incubated with streptavidin-horseradish peroxidase (HRP) for 1 hour at room temperature. Subsequently, the wells were washed 3 times with PB and a binding assay was performed using a TMB colorimetric substrate. The reaction was stopped with 0.1 N H2SO4, and the plates were quantitatively measured by optical density (A) at 450 nm on a Varioskan plate reader. As shown below, in Figures 3A and 3B, sICAM-1 significantly inhibited the binding of CD16a Fc receptor (P = 0.0039) and C1q (P = 0.0033), respectively. Similar results were observed for other antibodies tested using the same method. These data confirm the inhibitory effect of ICAM-1 on antibody humoral immune effector activity through physical binding. Example 2 - Identification of Key Residues Required for Binding to ICAM-1 and Generation of ICAM-1-Resistant IgG1-Type Antibodies

[0144] Strategies of IgG1 fragmentation and site-directed mutagenesis were employed to identify the ICAM-1 binding site or region. First, domain analysis of fragments was performed. Human IgG1 antibody was digested with papain to generate F(ab’)2 and Fc fragments, and purified by Protein A. F(ab’)2 was separated from the Protein A effluent, while the Fc fragment was captured and eluted by Protein A as previously described (Grasso L et al., Oncology Letters 23:2, 2022). The isolated fragments were quantified by Nanodrop (ThermoFisher) and equal amounts of fragments were used in ELISA format to localize sICAM-1 binding. Briefly, 100 μL of 2.5 μg / mL full-length IgG1 antibody or fragments were coated on the well plates in 0.05 M carbonate buffer at room temperature for 1 hour. The well plates were washed with phosphate buffered saline (PBS) plus 0.1% Tween-20 (PBS-T) and blocked with 200 μL of PBS plus 1% BSA. Subsequently, the wells were washed with PBS-T and incubated with 0.5 μg / mL biotinylated sICAM-1 in PBS plus 1% BSA on an orbital shaker at room temperature for 1 hour, repeated three times. The well plates were then washed three times with PBS-T and incubated a second time with 333 ng / mL streptavidin-HRP in PBS plus 1% BSA on an orbital shaker at room temperature for 1 hour. Then the well plates were washed three times with PBS-T and quantified using TMB colorimetric substrate (Pierce). The reaction was terminated with 0.1 N H2SO4 and A450 was quantified using a Varioskan plate reader. As shown below, in Figures 4A - 4B, sICAM-1 binds to the Fc domain (P = 0.00081, compared with HSA).

[0145] To further determine the binding position of sICAM-1 in the Fc domain, deletion mutagenesis analysis and ELISA competition assays were performed. A cDNA fusion construct containing the Ig signal peptide sequence and GST protein was synthesized, in which the IgG1 fragment was from the hinge region to the CH3 domain and carried a terminal FlagTag (Genscript), and it was subcloned into a eukaryotic expression plasmid driven by the CMV promoter. For deletion mutagenesis analysis, fragments were amplified from the full-length Fc construct using a 5' primer encoding HindIII and Kozak sequence, and a 3' primer encoding EcoRI and FlagTag. After separating each fragment, it was subcloned into the expression plasmid. For site-directed mutagenesis, the 5’ and 3’ regions were PCR amplified using overlapping primer pairs encoding codon changes. After purification, a second round of PCR was performed using flanking primers, and then the fragment was cloned into the expression plasmid. All constructs were subjected to DNA sequencing to confirm accuracy. The culture supernatant was analyzed by Western blotting using anti-FLAG antibody to ensure the correct molecular weight and quantification. The constructs were stably transfected into 293F cells, and the supernatant was harvested. The ability of the mutant proteins to compete with WT IgG1 was detected by ELISA.

[0146] In the ELISA competition experiment, the 96-well plate was coated with 2.5 μg / mL rituximab in 0.05 M carbonate buffer at room temperature for 1 hour. The plate was blocked as described above. After blocking, supernatants containing similar amounts of various expression fragments were added together with 0.5 μg / mL biotinylated or 1.5 μg / mL His-tagged full-length sICAM-1 (in triplicate), and incubated with shaking at room temperature for 1 hour. The plate was washed three times with PBS-T, and biotinylated ICAM-1 was detected with 333 ng / mL streptavidin-HRP, or anti-His-HRP (Sino Biologicals) diluted 1:3,000 in PBS containing 1% BSA and incubated with shaking at room temperature for 1 hour. Then the plate was washed three times with PBS-T, followed by the addition of TMB substrate for colorimetric quantitative reading. The reaction was stopped with 0.1 N H2SO4, and the A450 was quantitatively read using a Varioskan plate reader. Mutants that lost the ability to bind sICAM-1 could not compete for binding with the full-length sICAM-1 of rituximab coated on the plate and were determined to contain key residues. As outlined in Figure 5A and shown in Figures 5B and 5C, this domain and amino acids are located in the Kabat 407 to 410 region. Further analysis using amino acid substitutions within this region confirmed that these sequences are important for ICAM-1 binding, as determined by the competitive ELISA screening described above. As shown in Figure 8, additional amino acid substitution analysis unexpectedly revealed amino acid residues located near these motifs, such as Kabat 369–372 and Kabat 374– 377 may also have a negative impact on the binding of ICAM-1 to IgG1, indicating that modifications in the Kabat regions 369–410 can be used to create ICAM-1-tolerant antibodies and antibody-drug conjugates. Example 3 – Engineering of ICAM-1-tolerant IgG1 antibodies to enhance immune effector activity for maximal killing of tumor cells based on naked IgG1 therapy and antibody-drug conjugates (ADCs)

[0147] Based on the results described in Example 2, ICAM-1-tolerant IgG1-type antibodies can now be developed by engineering an IgG1 Fc domain that is tolerant to ICAM-1 binding to maximize immune effector activity and potentially develop antibody-drug conjugates (ADCs). As described above, isogenic cells in which ICAM-1 protein is knocked down or knocked out can be used to screen for IgG1 mutations within or near the IC1 region. Additionally, an IgG1 heavy chain cassette modified as shown in Figure 6 can be generated, or any one, two, three, or four amino acid substitutions can be made, controlling amino acid substitutions within the range of SEQ ID NO3 (or close, e.g., Kabat 369-372 or Kabat 374-377), allowing for the development of enhanced IgG1 molecules with improved immune effector activity and ADCs that are not hampered by reduced ICAM-1 binding and internalization rates, parameters that are crucial for maximal killing of ADC target cells.

[0148] In the presence of ICAM-1 positivity in cancer, the use of IC1-modified IgG1 is very useful. These cancers include, but are not limited to, lymphoma, multiple myeloma, breast cancer, gastric cancer, head and neck cancer, and colorectal cancer. Patients can first be screened to determine their ICAM-1 status, and patients with elevated ICAM-1 expression detected by immunohistochemistry or serum ELISA that can monitor sICAM-1 above the normal range by 5% can benefit from IC1-modified antibodies. These include, but are not limited to, IC1-modified rituximab (SEQ ID NO: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), in the presence of sICAM-1, showed significantly enhanced ADCC activity compared to parental rituximab (RTX) using the Jurkat-CD16a reporter cell line and Daudi target cell assay as described in Example 1, P = 0.0015. These results support the use of modified antibodies for the treatment of diseases involving overexpression of the immunosuppressive ICAM-1 protein.

[0149] All references cited herein are expressly incorporated herein by reference. Amino acid sequence Sequence identifier: 1 (mature membrane-bound human ICAM-1 / CD54) QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPP Sequence identifier: 2 (soluble human ICAM-1 / CD54) QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYE Sequence identifier: 3407 - 410 YSKL Sequence identifier: 4 (Rituximab heavy chain, IC1 region in bold and underlined) Sequence identifier: 5 (Rituximab - N109D, heavy chain with IC1 region and CA125 - resistant residues in bold and underlined) Sequence identifier: 6 (Trastuzumab heavy chain, IC1 region in bold and underlined) Sequence identifier: 7 (Pertuzumab heavy chain, IC1 region in bold and underlined) Sequence identifier: 8 (Cetuximab heavy chain, IC1 region in bold and underlined) Sequence identifier: 9 (Daratumumab heavy chain, IC1 region in bold and underlined) Sequence identifiers: 10, 11, 12, 13 (ICAM - 1 shRNAs) shRNA 312270A 1 GGATCGCACTGTGGTAGCAGCCGCAGTCATAATGGTCAAGAGCCATTATGACTGCGGCTGCTACCACAGTGTTTTTTGAAGCTT shRNA 312270B 2 GGATCGTACCTCTATAACCGCCAGCGGAAGATCAATCAAGAGTTGATCTTCCGCTGGCGGTTATAGAGGTATTTTTTGAAGCTT shRNA 312270C 3 GGATCGACCTTCCTCACCGTGTACTGGACTCCAGATCAAGAGTCTGGAGTCCAGTACACGGTGAGGAAGGTTTTTTTGAAGCTT shRNA 312270D 4 GGATCGATTTCTCGTGCCGCACTGAACTGGACCTGTCAAGAGCAGGTCCAGTTCAGTGCGGCACGAGAAATTTTTTTGAAGCTT

Claims

1. An immunosuppressive-tolerant human IgG1 antibody, which comprises one to four amino acid substitutions relative to an immunosuppressive-sensitive human IgG1 antibody without one to four amino acid substitutions, wherein the immunosuppression of the immunosuppressive-sensitive human IgG1 is caused by soluble or membrane-bound ICAM-1 / CD54.

2. The immunosuppressive-tolerant human IgG1 antibody according to claim 1, wherein the antibody is a full-length antibody.

3. The immunosuppressive-tolerant human IgG1 antibody according to claim 1, wherein the antibody is an antibody-drug conjugate.

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

1.

5. The immunosuppressive-tolerant human IgG1 antibody according to claim 1, wherein the ICAM-1 / CD54 is soluble ICAM-1 / CD54, such as SEQ ID NO:

2.

6. The immunosuppressive-tolerant human IgG1 antibody according to claim 1, wherein the one to four amino acid substitutions are located within Kabat residues 367-425.

7. The immunosuppressive-tolerant human IgG1 antibody as claimed in claim 1, wherein the 1 to 4 amino acid substitutions are located within Kabat residues 369-410.

8. The immunosuppressive-tolerant human IgG1 antibody as claimed in claim 6, wherein the 1 to 4 amino acid substitutions are located within the YSKL motif (sequence ID number: 3) in the heavy chain of the immunosuppressive-tolerant human IgG1 antibody.

9. The immunosuppressive-tolerant human IgG1 antibody as claimed in claim 6, which comprises an IgG1 light chain and an IgG1 heavy chain.

10. The immunosuppressive-tolerant human IgG1 antibody according to claim 1, which is conjugated to a drug.

11. A polynucleotide encoding the immunosuppressive-tolerant human IgG1 antibody as claimed in claim 1.

12. A nucleic acid vector encoding the immunosuppressive-tolerant human IgG1 antibody as claimed in claim 1.

13. A stable cell line comprising the nucleic acid vector according to claim 12, wherein the stable cell line expresses the immunosuppressive-tolerant human IgG1 antibody.

14. A method for treating a patient suffering from a disease, wherein the patient expresses an elevated level of ICAM-1 / CD54 compared to a healthy human population, the method comprising: administering to the patient the immunosuppressive-tolerant human IgG1 antibody according to claim 1.

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

16. The method according to claim 14, wherein the disease is an inflammatory disease.

17. The method according to claim 15, wherein the cancer includes Hodgkin lymphoma, non-Hodgkin 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. A method for treating a cancer patient or an inflammatory disease patient, comprising: Administering a full-length human IgG1 antibody to a cancer patient or a patient with an inflammatory disease, said antibody comprising a heavy chain having one to four amino acid substitutions in the motif YSKL (SEQ ID NO:3) of the full-length human IgG1 antibody.

19. The method according to claim 18, wherein the full-length human IgG1 antibody comprises the light chain of rituximab and binds to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

4.

20. The method according to claim 19, wherein the full-length human IgG1 antibody comprises the light chain of rituximab-N109D and binds to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

5.

21. The method according to claim 18, wherein the full-length human IgG1 antibody comprises the light chain of trastuzumab and binds to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

6.

22. The method according to claim 18, wherein the full-length human IgG1 antibody comprises the light chain of pertuzumab and binds to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

7.

23. The method according to claim 18, wherein the full-length human IgG1 antibody comprises the light chain of cetuximab bound to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

8.

24. The method according to claim 18, wherein the full-length human IgG1 antibody comprises the light chain of daratumumab bound to an immunosuppressive tolerance heavy chain, such as SEQ ID NO:

9.

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

26. The method according to claim 21 or 22, wherein the patient is a cancer patient and the patient has a cancer of breast cancer, gastric cancer or head and neck cancer.

27. The method according to claim 23, wherein the patient is a cancer patient and the patient has a cancer selected from colorectal cancer and head and neck cancer.

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

29. The method according to 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 a homologous cell line with reduced or no expression of ICAM-1 / CD54.

30. The method according to 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. The method according to claim 29, wherein the homologous second human cancer cell line with reduced or no expression of ICAM-1 / CD54 comprises shRNA 1-4 (SEQ ID NO: 10-13).

32. The method according to 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. A method for screening a candidate immunosuppressive tolerance human IgG1 antibody that has one to four amino acid substitutions relative to an immunosuppressive sensitive human IgG1 antibody to identify a candidate antibody refractory to ICAM-1 / CD54 immunosuppression, comprising: (a) contacting the candidate immunosuppressive tolerance human IgG1 antibody with biotinylated human CD16a Fc receptor, wherein the contact is carried out in the presence of sICAM-1 / CD54, as shown below: SEQ ID NO: 2; (b) contacting the immunosuppressive sensitive human IgG1 antibody with biotinylated human CD16a Fc receptor, wherein the contact is carried out in the presence of sICAM-1 / CD54, as shown below: SEQ ID NO: 2; and (c) determining the binding of the antibodies in steps (a) and (b) to the CD16a Fc receptor.

34. A method for screening a candidate immunosuppressive tolerance human IgG1 antibody that has one to four amino acid substitutions relative to an immunosuppressive susceptible human IgG1 antibody to identify a candidate antibody tolerant to ICAM-1 / CD54 immunosuppression, the method comprising: (a) contacting the candidate immunosuppressive tolerance human IgG1 antibody with biotinylated human C1q protein, wherein the contact is carried out in the presence of sICAM-1 / CD54, as shown below: SEQ ID NO: 2; (b) contacting the immunosuppressive susceptible human IgG1 antibody with biotinylated human C1q protein, wherein the contact is carried out in the presence of sICAM-1 / CD54, as shown below: SEQ ID NO: 2; and (c) determining the binding of the antibodies in steps (a) and (b) to the biotinylated human C1q protein.

35. A method for screening a candidate immunosuppressive tolerance human IgG1 antibody that has one to four amino acid substitutions relative to an immunosuppressive sensitive human IgG1 antibody in the form of an antibody-drug conjugate to identify a candidate tolerant to ICAM-1 / CD54 immunosuppression, comprising: (a) Contact a first target cell expressing ICAM-1 / CD54 and a target antigen with a candidate immunosuppressive tolerant human IgG1 antibody; (b) Contact a second target cell not expressing ICAM-1 / CD54 but expressing the target antigen with the candidate immunosuppressive tolerant human IgG1 antibody; wherein the first and second target cells are syngeneic; and (c) Compare the amount of cell death caused by the candidate immunosuppressive tolerant human IgG1 antibody in steps (a) and (b).

36. A method for identifying a tumor-bearing patient suitable for treatment with an antibody-drug conjugate, comprising: Detecting each tumor in a plurality of patients and determining whether each tumor in the plurality of patients expresses ICAM-1; If the tumor does not express ICAM-1, recommending treatment of the patient with an IgG1 antibody-drug conjugate, wherein the IgG1 antibody is sensitive to immunosuppression; and If the tumor expresses ICAM-1, recommending not to treat the patient with an IgG1 antibody-drug conjugate, wherein the IgG1 antibody is sensitive to immunosuppression; and Optionally treating at least one patient not expressing ICAM-1 with an IgG1 antibody-drug conjugate, wherein the IgG1 antibody is sensitive to immunosuppression.

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