Human anti-CD33 antibody and its use

Abstract

We provide a novel anti-CD33 antibody. [Solution] The antibody provided is a pan-conjugate that binds to the C2-set Ig-like domain of CD33 in the presence or absence of the V-set Ig-like domain, or a V-set conjugate that binds to the V-set Ig-like domain of CD33. This antibody provides a novel therapeutic and diagnostic means for CD33-related disorders such as acute myeloid leukemia (AML).

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 003,203, filed on 31 March 2020, which is incorporated herein by reference in its entirety as if it were fully contained herein.

[0002] Statement on federally supported research or development This invention was made with government support under CA234203 and CA223409 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Sequence listing reference The sequence listing relating to this application is provided in text format instead of being a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 2GU_ST25.txt. The text file is 196KB in size, was created on March 31, 2021, and filed electronically via EFS-Web.

[0004] Areas of this disclosure A set of novel anti-CD33 antibodies is described. The antibodies provided are pan-conjugates that bind to the C2-set Ig-like domain of CD33 in the presence or absence of the V-set Ig-like domain, C2-set conjugates that bind to the C2-set Ig-like domain only in the absence of the V-set Ig-like domain of CD33, or V-set conjugates that bind to the V-set Ig-like domain of CD33. These antibodies provide novel therapeutic and diagnostic tools for CD33-related disorders such as acute myeloid leukemia (AML). [Background technology]

[0005] According to the World Health Organization, cancer is the second leading cause of death globally, accounting for an estimated 9.6 million deaths in 2018. Acute myeloid leukemia (AML) is a type of cancer caused by malignant tumors of clonal proliferative myeloblastic cells. In the United States, there are 20,000 new cases of AML annually, and 11,000 people die from AML each year (Siegel et al., 2021, CA Cancer J Clin. 71(1): pp. 7-33). While young patients with AML can achieve high complete remission rates of 60% to 80% with conventional chemotherapy (Dohner et al., 2017, Blood. 129(4): pp. 424-447), treatment outcomes for elderly patients aged 65 and over remain inadequate, with as many as 70% dying from the disease within one year of diagnosis (Meyers et al., Appl Health Economic Health Policy, 11: pp. 275-286, 2013). Unfortunately, due to the chemotherapy resistance of leukemia stem cells, relapse after conventional treatment is common (Eppert et al., 2011. Nat. Med. 17(9): pp. 1086-1093), and current treatment options for relapsed / refractory (R / R) AML are poor, resulting in a 12-month overall survival rate of less than 30%.

[0006] CD33 is a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) protein family. It is a 67 kDa glycosylated transmembrane protein. CD33 (also known as SIGLEC-3) is a myeloid differentiation antigen found in at least some leukemia cells, and possibly in some cases in AML stem cells, in almost all patients with AML. Based on this widespread expression pattern, CD33 has been widely pursued as a therapeutic target in AML. Recent data from several randomized studies have demonstrated that gemtuzumab ozogamicin (GO), a CD33 antibody-drug conjugate, improves survival when added to chemotherapy in a defined subset of patients with newly diagnosed AML. This data confirms that CD33 is the first (and so far only) target for immunotherapy in AML. In parallel with the development of new and more effective CD33-targeted therapies (e.g., antibody-drug conjugates, radioactive immunoconjugates, bispecific antibodies, chimeric antigen receptor [CAR]-modified T cells) to overcome the limitations pointed out by GO, there is growing interest in CD33 as a drug target for other malignant and non-malignant disorders. These efforts include targeting CD33 splice variants not recognized by GO, as well as targeting CD33+ tumor cells in other hematological malignancies, CD33+ myeloid-derived suppressor cells (MDSCs) in various diseases, and normal CD33+ microglia in Alzheimer's disease (Walter, Expert Opin Biol Ther. 2020, 20(9):955-958).

[0007] Full-length CD33 protein (CD33 FL CD33 is characterized by a V-set immunoglobulin (Ig)-like domain at the distal membrane of the amino terminus and a C2-set Ig-like domain at the proximal membrane in its extracellular portion (Figure 1). Shorter isoforms of CD33 exist. These shorter isoforms of CD33 have a V-set domain (CD33 ΔE2It includes one variant lacking exon 2, which codes for ). At least at the mRNA level, CD33 ΔE2 CD33 is widely expressed in bone marrow and peripheral blood myeloid cells of patients with AML. However, almost all commercially and clinically available CD33 antibodies currently recognize the immunodominant V-set Ig-like domain. This means that these antibodies do not recognize CD33 ΔE2 This means that shorter forms of CD33, such as those lacking the V-set domain, will not be recognized. ΔE2 Selective transcription and CD33 FL This can explain the observation made in one clinical trial in pediatric AML that patients with a single nucleotide polymorphism in the CD33 gene, resulting in reduced translation, did not benefit from the addition of GO (which also binds to the V-set domain of CD33) to intensive chemotherapy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Siegel et al., 2021, CA Cancer J Clin.71(1):7-33 [Non-Patent Document 2] Dohner et al., 2017. Blood. 129(4): 424-447. [Non-Patent Document 3] Meyers et al., Appl Health Economic Health Policy, 11: pp. 275-286, 2013. [Non-Patent Document 4] Eppert et al., 2011. Nat. Med. 17(9): 1086-1093. [Non-Patent Document 5] Walter, Expert Opin Biol Ther. 2020, 20(9):955-958. [Overview of the project] [Means for solving the problem]

[0009] (Summary of the present disclosure) The present disclosure provides antibodies that bind / recognize C2-set Ig-like domains in CD33 protein, regardless of the presence of V-set Ig-like domains (Figure 1). These antibodies are referred to as pan-binding agents (CD33 PAN antibodies). Pan-binding agents can bind to target cells expressing shorter isoforms such as CD33 FL and CD33 ΔE2 variants, etc., and thus can target a higher percentage of CD33-expressing cells. The CD33 PAN antibodies disclosed herein include 1H10, 1A9, 1E6, 1D2, and 1B9.

[0010] The present disclosure also provides newly developed anti-CD33 antibodies that bind to the V-set Ig-like domain of CD33. These V-set binding agents include 1H8, 2D3, and 2E3, and provide additional diagnostic and treatment options for patients expressing CD33 FL .

[0011] <( The antibodies disclosed herein can be engineered into numerous formats such as anti-CD33 antibody conjugates. Anti-CD33 antibody conjugates are artificial molecules that include a molecule conjugated to a CD33 antibody-based binding domain. Anti-CD33 antibody conjugates include anti-CD33 immunotoxins, antibody-drug conjugates (ADCs), and radioisotope conjugates. The antibodies disclosed herein can also be engineered into anti-CD33 multispecific antibodies (e.g., anti-CD33 bispecific antibodies, anti-CD33 trispecific antibodies, anti-CD33 quadrispecific antibodies, etc.). In the case of the multispecific format, the engineered molecule can bind to CD33 as well as to immunoreactivation epitopes on immune cells such as, for example, CD3, CD16, CD28, CD64, and / or 4-1BB. These embodiments bring activated immune cells to CD33-expressing cells and help destroy CD33-expressing cells.

[0012] Some of the drawings submitted herein are better understood in color. The applicant considers the color versions of the drawings to be part of the original submission and reserves the right to present color images of the drawings in subsequent proceedings. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows CD33 (CD33ΔE2) with exon 2 deletion, resulting in the deletion of the full-length CD33 (CD33FL) and the V set domain. Antibodies that bind to CD33FL alone (anti-CD33FL), CD33ΔE2 alone (anti-CD33ΔE2), or CD33FL and CD33ΔE2 (anti-CD33FL+ΔE2 or anti-CD33PAN) are shown. [Figure 2] This is a schematic diagram of an artificial CD33 molecule (CD33ΔE3-4) with deletions of exons 3 and 4 resulting in membrane-proximal rearrangement of the CD33FL and V set domains, or an insertion of either two C2 set domains of CD22 ("CD33FL+CD22 2D") or four C2 set domains of CD22 ("CD33FL+CD22 4D"). CD33ΔE3-4 was engineered using site-directed mutagenesis to splice out CD33 amino acids (aa) 140-232 of the human CD33FL extracellular domain (ECD). CD33FL+CD22 4D was generated using endogenous CD33 signal peptides (aa1-17), a 6-histidine tag, a 3× glycine linker, human CD33 ECD (aa18-259), a portion of human CD22 ECD containing C2 type domains 3-6 (aa331-683), the CD33 transmembrane domain, and the CD33 intracellular domain (aa260-364). CD22 aa331-504 (C2 type domains 3 and 4) were removed from CD33FL+CD22 24 to generate CD33FL+CD22 2D. [Figure 3A]This figure shows that the antitumor effect of CD33 / CD3 BsAb against human myeloid leukemia cells is enhanced by a decrease in the binding distance from the cell membrane. Human CD33+ myeloid leukemia cell lines ((3A)ML-1, (3B)HL-60, (3C)K562) with CRISPR / Cas9-mediated deletion of the endogenous CD33 locus were engineered to overexpress either CD33FL or CD33ΔE3-4 via lentiviral gene transfer. Relative expression of the target protein was assessed by flow cytometry with the V-set domain CD33 antibody, P67.6, and representative histograms are shown in the lower right panel. Cells were then treated with V-set domain targeting CD33 / CD3 BsAb at a concentration of 1000 pg / mL and healthy donor T cells enriched from unstimulated peripheral blood mononuclear cells collected from healthy adult volunteers with the shown effector:target (E:T) cell ratio (upper panel). Bone marrow cells were also treated with gemtuzumab ozogamicin (GO) at the indicated concentrations (lower left panel). Cytotoxicity was quantified by flow cytometry as the change in the percentage of dead cells measured by 4',6-diamidino-2-phenylindole (DAPI) staining at 2 days (for BsAb) or 3 days (for GO). [Figure 3B]This figure shows that the antitumor effect of CD33 / CD3 bispecific antibodies (BsAbs) against human myeloid leukemia cells is enhanced by a decrease in the binding distance from the cell membrane. Human CD33+ myeloid leukemia cell lines ((3A)ML-1, (3B)HL-60, (3C)K562) with CRISPR / Cas9-mediated deletion of the endogenous CD33 locus were engineered to overexpress either CD33FL or CD33ΔE3-4 via lentiviral gene transfer. The relative expression of the target protein was evaluated by flow cytometry using the V-set domain CD33 antibody, P67.6, and representative histograms are shown in the lower right panel. The cells were then treated with a V-set domain-targeting CD33 / CD3 BsAb at a concentration of 1000 pg / mL and healthy donor T cells enriched from unstimulated peripheral blood mononuclear cells collected from healthy adult volunteers with the indicated effector:target (E:T) cell ratio (upper panel). Bone marrow cells were also treated with gemtuzumab ozogamicin (GO) at the indicated concentration (lower left panel). Cytotoxicity was quantified by flow cytometry as the change in the percentage of dead cells measured by 4',6-diamidino-2-phenylindole (DAPI) staining at 2 days (for BsAb) or 3 days (for GO). [Figure 3C]This figure shows that the antitumor effect of CD33 / CD3 bispecific antibodies (BsAbs) against human myeloid leukemia cells is enhanced by a decrease in the binding distance from the cell membrane. Human CD33+ myeloid leukemia cell lines ((3A)ML-1, (3B)HL-60, (3C)K562) with CRISPR / Cas9-mediated deletion of the endogenous CD33 locus were engineered to overexpress either CD33FL or CD33ΔE3-4 via lentiviral gene transfer. The relative expression of the target protein was evaluated by flow cytometry using the V-set domain CD33 antibody, P67.6, and representative histograms are shown in the lower right panel. Cells were then treated with a V-set domain-targeting CD33 / CD3 BsAb at a concentration of 1000 pg / mL and healthy donor T cells enriched from unstimulated peripheral blood mononuclear cells collected from healthy adult volunteers with the indicated effector:target (E:T) cell ratio (upper panel). Bone marrow cells were also treated with gemtuzumab ozogamicin (GO) at the indicated concentration (lower left panel). Cytotoxicity was quantified by flow cytometry as the change in the percentage of dead cells measured by 4',6-diamidino-2-phenylindole (DAPI) staining at 2 days (for BsAb) or 3 days (for GO). The anti-V-set domain-targeting CD33 / CD3 BsAb was constructed in scFv-scFv format using constructs referred to herein as RC1 or A3, utilizing the sequence shown in Sequence ID No. 259 and described in U.S. Patent Application Publication No. 2016 / 0317657. *p<0.05;**p<0.01;***p<0.001. [Figure 4]This figure shows that the antitumor effect of CD33 / CD3 BsAb on human acute lymphoblastic leukemia cells engineered to express the CD33 protein is enhanced by a reduction in the binding distance from the cell membrane. Human CD33neg acute lymphoblastic leukemia (ALL) cell line RS4;11 was engineered to overexpress either CD33FL or CD33ΔE3-4 via lentiviral gene transfer. Relative expression of the target protein was assessed by flow cytometry using the V-set domain CD33 antibody, P67.6, and representative histograms are shown in the lower panel. Cells were then treated with a concentration of 1000 pg / mL of V-set domain targeting CD33 / CD3 BsAb and healthy donor T cells enriched from unstimulated peripheral blood mononuclear cells collected from healthy adult volunteers with the indicated effector:target (E:T) cell ratio (upper panel). Cytotoxicity was quantified by flow cytometry as the change in the percentage of dead cells measured by DAPI staining after 2 days. The anti-V set domain-directed CD33 / CD3 BsAb was constructed in scFv-scFv format using constructs referred to herein as RC1 or A3, utilizing the sequence shown in Sequence ID No. 259 and described in U.S. Patent Application Publication No. 2016 / 0317657. *p<0.05; **p<0.01; ***p<0.001. [Figure 5]This figure shows that the antitumor effect of CD33 / CD3 BsAb decreases with increasing binding distance from the cell membrane. Human myeloid leukemia cell lines with CRISPR / Cas9-mediated deletion of the endogenous CD33 locus (ML-1 [upper panel], K562 [lower panel]) were engineered to overexpress CD33FL, CD33FL+CD22 2D, or CD33FL+CD22 4D via lentiviral gene transfer. Relative expression of CD33 constructs was assessed by flow cytometry using the V-set domain CD33 antibody, P67.6 (right panel). Cells were then treated with V-set domain targeting CD33 / CD3 BsAb at the indicated concentrations (pg / mL) and healthy donor T cells enriched from healthy donor unstimulated peripheral blood mononuclear cells with a 1:1 E:T cell ratio. Cytotoxicity was quantified by flow cytometry as the change in the percentage of dead cells measured by DAPI staining after 2 days. The anti-V set domain-directed CD33 / CD3 BsAb was constructed in scFv-scFv format using constructs referred to herein as RC1 or A3, utilizing the sequence shown in Sequence ID No. 259 and described in U.S. Patent Application Publication No. 2016 / 0317657. *p<0.05; **p<0.01; ***p<0.001. [Figure 6A] Human CD33PAN antibody clones (clones 1A9, 1H10, 1B9, 1E6, and 1D2) were tested by flow cytometry as directed against CD33+ parental ML-1 cells and ML-1 cells with CRISPR / Cas9-mediated deletion of CD33 ("CD33 KO"), as well as against REH sublines engineered to express CD33FL or CD33ΔE2. A control without primary antibody was included. [Figure 6B] Human CD33V set antibody clones (clones 2E3, 2D3, and 1H8) were tested by flow cytometry as directed against CD33+ parental ML-1 cells and ML-1 cells with CRISPR / Cas9-mediated deletion of CD33 ("CD33 KO"), as well as against REH sublineages engineered to express CD33FL or CD33ΔE2. A control without primary antibody was included. [Figure 7A]This figure shows the reaction rate profile established from anti-CD33 antibody purified using surface plasmon resonance (SPR) technology on a Carterra instrument. SPR is an excellent method for estimating the reaction rate constant of the binding interaction, which can be fitted into a 1:1 Langmuir binding model to determine the on-rate (ka) and off-rate (kd). Both of these rate parameters allow for the calculation of the dissociation rate constant (kD), referred to as binding affinity. Antibody clones were captured as an array on a protein A / G loan, which was immobilized on an HC30M tip. The first kinetic experiment used full-length CD33 (CD33FL) antigen, which was started at a concentration of 2 μM prior to 4-fold titration up to 2 nM. After 10 HBSTE buffer blanks, six injections from low to high concentrations were subsequently flowed onto the array to evaluate the reaction rate of each clone printed on the array: 1 min baseline, 5 min association, and 10 min dissociation. Next, if any antigens remained bound to the array, preventing interaction with the second antigen of interest, the chip was regenerated in 0.85% phosphate pH 1.7 for a new reprint of the same array cloned onto a protein A / G loan, in which CD33ΔE2 was flowed onto the antibody array. Carterra reaction rate software was used to process the data to fit the raw data into reaction rate curves for each concentration of antigen injected onto the array. [Figure 7B-1]SPR evaluation of purified ECD from captured 1H10 and 2D3-bound CD33FL or CD33ΔE2. Experiments were performed on a Biacore T100 apparatus using Series S CM4 tips at 25°C. 60 μg / mL of protein A / G in 10 mM sodium acetate at pH 4.0 was immobilized on two flow cells (1000 RU) using a standard amine coupling reaction. Capture kinetics experiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, and 0.1 mg / mL IgG-free bovine serum albumin buffer. 0.5 μg / mL anti-human CD33 antibody was injected at 10 μL / min onto a second flow cell of immobilized protein A / G for 30–40 seconds to capture 40–58 RU of antibody in CD33FL binding experiments, or for 45–80 seconds to capture 70–95 RU of antibody in CD33ΔE2 binding experiments. Purified external domains for CD33FL and CD33ΔE2 were flowed as a concentration series at 50 μL / min onto both the captured antibody and protein A / G alone (reference) surfaces. The CD33FL series was started at a high concentration of 160 nM for 2D3 and 1H10, while the CD33ΔE2 series was started at 40 nM for 1H10 and 300 nM for 2D3. CD33 was injected for 7 minutes and allowed to dissociate for 20 or 30 minutes for most pairs. Two-fold serial dilutions of the external domain concentration were performed randomly in two different ways, with a buffer blank included every four injections. CM4 tips were regenerated with two 30-second injections of 0.85% H3PO4 at 50 μL / min, and the antibody was recaptured prior to each CD33 injection. Data were double-referenced and analyzed in BiaEval 2.0.4 software using a 1:1 binding model with local Rmax. [Figure 7B-2]SPR evaluation of purified ECD from captured 1H10 and 2D3-bound CD33FL or CD33ΔE2. Experiments were performed on a Biacore T100 apparatus using Series S CM4 tips at 25°C. 60 μg / mL of protein A / G in 10 mM sodium acetate at pH 4.0 was immobilized on two flow cells (1000 RU) using a standard amine coupling reaction. Capture kinetics experiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, and 0.1 mg / mL IgG-free bovine serum albumin buffer. 0.5 μg / mL anti-human CD33 antibody was injected at 10 μL / min onto a second flow cell of immobilized protein A / G for 30–40 seconds to capture 40–58 RU of antibody in CD33FL binding experiments, or for 45–80 seconds to capture 70–95 RU of antibody in CD33ΔE2 binding experiments. Purified external domains for CD33FL and CD33ΔE2 were flowed as a concentration series at 50 μL / min onto both the captured antibody and protein A / G alone (reference) surfaces. The CD33FL series was started at a high concentration of 160 nM for 2D3 and 1H10, while the CD33ΔE2 series was started at 40 nM for 1H10 and 300 nM for 2D3. CD33 was injected for 7 minutes and allowed to dissociate for 20 or 30 minutes for most pairs. Two-fold serial dilutions of the external domain concentration were performed randomly in two different ways, with a buffer blank included every four injections. CM4 tips were regenerated with two 30-second injections of 0.85% H3PO4 at 50 μL / min, and the antibody was recaptured prior to each CD33 injection. Data were double-referenced and analyzed in BiaEval 2.0.4 software using a 1:1 binding model with local Rmax. [Figure 8] Internalization of 1E6 and P67.6. AML cell lines were incubated with CD33 antibody at 37°C for the indicated time. A fluorescently labeled secondary antibody was then added to quantify the CD33 antibody remaining on the cell surface. Results are presented as the percentage of fluorescence signal present at time 0. [Figure 9]Binding of recombinant fully human CD33V set antibody 1H8 with a human IgG1 framework to REH cells (human acute lymphoblastic leukemia cell line, endogenously CD33neg) engineered to express human CD33FL. [Figure 10] Human CD33PAN / CD3 BsAb re-induces T cell-mediated cytotoxicity in human CD33+ AML cells. Parental AML cell lines were treated with healthy donor T cells using the indicated effector:target (E:T) cell ratio and various doses of 1E6 / CD3 BsAb. Cytotoxicity was quantified by flow cytometry at 2 days as the change in percentage of dead cells, measured by DAPI staining. The CD33rs12459419 genotype is shown in parentheses. [Figure 11] Human CD33PAN / CD3 BsAb specifically re-induces T cell-mediated cytotoxicity in human AML cells. Parental ML-1 cells and sublineages in which CD33 was CRISPR / Cas9-mediated knockout (KO) were treated with 1E6 BaAb at the indicated concentrations and healthy donor T cells with a 1:1 E:T ratio. Dead leukemia cells were counted by flow cytometry after 48 hours, and the change in dead cells compared to no BsAb treatment is shown. Mean ± SEM values ​​from three separate experiments are shown. ***p<0.001;****p<0.0001. [Figure 12] This figure shows how human CD33PAN / CD3 BsAb re-induces T cell-mediated cytotoxicity against human acute leukemia cells in a CD33 and epitope-specific manner. Parental CD33neg REH cells or sublines engineered to overexpress CD33FL or CD33ΔE2 were treated with anti-V set CD33 / CD3 BsAb or 1E6 BsAb at a dose of 1000 pg / mL and a 3:1 E:T ratio. Dead leukemia cells were counted by flow cytometry after 48 hours, and the change in dead cells compared to no BsAb treatment is shown. Mean ± SEM values ​​from three separate experiments are shown. [Figure 13]This figure shows that human CD33PAN / CD3 BsAb re-induces T cell-mediated cytotoxicity against primary human AML cells. A panel of 11 primary AML patient samples was treated with 1E6 / CD3 BsAb and healthy donor T cells with the indicated E:T ratio. Cytotoxicity was determined after 2 days by flow cytometry, counting both dead cells (using 4',6-diamidino-2-phenylindole [DAPI] staining) and total cell count. Mean cytotoxicity ± SEM across the 11 patient samples is shown. [Figure 14-1]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-2]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-3]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-4]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-5]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-6]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-7]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-8]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-9]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-10]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-11]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-12]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-13]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Figure 14-14]Sequences supporting the disclosure: Human full-length (FL) CD33 with a mouse Fc domain, used as an immunogen against human FLCD33 (hsCD33-mmFc; SEQ ID NO: 2); Human ΔE2 version of CD33 with a mouse Fc domain (CD33ΔE2), used as an immunogen against human CD33ΔE2 (hsCD33_ΔE2-mmFc; SEQ ID NO: 3); Mouse protein ECD (SEQ ID NO: 4) and coded (SEQ ID NO: 5) CD33 in which the mouse-derived C2 set Ig-like domain is replaced with a human CD33-derived C2 set Ig-like domain, combined with the human IgG1-derived Fc region of CD33. Used as an immunogen to produce antibodies against the 2-set domain (mmCD33_V-set-mmCD33_C2-set-hsCD33_Fc_hslgG1); mouse protein ECD (SEQ ID NO: 6) and coded (SEQ ID NO: 7) CD33, in which the mouse-derived C2-set Ig-like domain is replaced with a human CD33-derived C2-set Ig-like domain, both combined with human CD33-derived transmembrane domains and cleaved intracellular domains, and used as an immunogen to produce antibodies against the human C2-set domain of CD33; and full-length human protein (SEQ ID NO: 8) and coded (SEQ ID NO: 9) CD33 Immunogens for human CD33; human ΔE2 protein (SEQ ID NO: 10) and coding sequence (SEQ ID NO: 11); CD33 (CD33ΔE2), immunogens for human CD33ΔE2); CD33:CD22 4D protein (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 135); CD33:CD22 2D protein (SEQ ID NO: 136) and coding sequence (SEQ ID NO: 137); CD33 V-set construct (exon 3 and 4 deletion) protein (SEQ ID NO: 138) and coding sequence (SEQ ID NO: 139); CD33 signal peptide (SEQ ID NO: 140) and coding sequence (SEQ ID NO: 141); 6-histidine tag (SEQ ID NO: 142) and coding sequence (SEQ ID NO: 143); 3× glycine linker and coding sequence; CD33 ECD (SEQ ID NO: 145) and coding sequence (SEQ ID NO: 146); CD33 lacking amino acids 140-232 ECD (SEQ ID NO: 147) and coding sequence (SEQ ID NO: 148); CD33 transmembrane domain (SEQ ID NO: 149) and coding sequence (SEQ ID NO: 150);CD33 intracellular domain (SEQ ID NO: 151) and coding sequence (SEQ ID NO: 152); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and Ig-like C2-type 6 (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 154); a portion of CD22 ECD containing CD22 domains defined as Ig-like C2-type 5 and Ig-like C2-type 6 (SEQ ID NO: 155) and coding sequence (SEQ ID NO: 156); 1E6 / CD3 bispecific molecule (SEQ ID NO: 157); IgK signal peptide (SEQ ID NO: 158); 1H10 scFv VH-VL orientation (SEQ ID NO: 230); 1H10 scFv VL-VH orientation (SEQ ID NO: 231); 1A9 scFv VH-VL orientation (SEQ ID NO: 232); 1A9 scFv VL-VH orientation (SEQ ID NO: 233); 1E6 scFv VH-VL orientation (SEQ ID NO: 234); 1E6 scFv VL-VH orientation (SEQ ID NO: 235); 2D3 scFv VH-VL orientation (SEQ ID NO: 236); 2D3 scFv VL-VH orientation (SEQ ID NO: 237); 1H10 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 238); 1H10 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 239); 1A9 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 240); 1A9 scFv VL-VH orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 241); 1E6 scFv VH-VL orientation, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 242); 1E6 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 243); 2D3 scFv VH-VL oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 244); 2D3 scFv VL-VH oriented, bispecific antibody CD33 / CD3 engager (SEQ ID NO: 245); Human CD33 full-length DNA code (used for cell-based immunogens; CD33 signal peptide code sequence in bold) (SEQ ID NO: 246); Human CD33 full-length protein (SEQ ID NO: 247);Also, 1H10, 1A9, 1E6, and / or 1B9 light chain signal peptides (SEQ ID NO: 248); 1D2 light chain signal peptide (SEQ ID NO: 249); 1H8 light chain signal peptide (SEQ ID NO: 250); 2D3 light chain signal peptide (SEQ ID NO: 251); 1H10 heavy chain signal peptide (SEQ ID NO: 252); 1A9 heavy chain signal peptide (SEQ ID NO: 253); 1E6 and / or 2E3 heavy chain signal peptide (SEQ ID NO: 254); 1D2 heavy chain signal peptide (SEQ ID NO: 255); 1B9 heavy chain signal peptide (SEQ ID NO: 256); 1H8 heavy chain signal peptide (SEQ ID NO: 257); 2D3 heavy chain signal peptide (SEQ ID NO: 258); V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 259); and V-set oriented CD33 / CD3 BsAb(RC1) (SEQ ID NO: 260) without a reader sequence or His tag. [Modes for carrying out the invention]

[0014] According to the World Health Organization, cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Acute myeloid leukemia (AML) is a malignant tumor of clonal proliferative myeloblastic cells. AML is also known as acute myeloid leukemia, acute myeloid leukemia, acute granulocytic leukemia, and acute nonlymphoblastic leukemia.

[0015] High complete remission rates for patients with AML can be achieved with conventional chemotherapy in 60%–80% of young adults and 40%–60% of adults over 60 years of age (Dohner et al., 2017. Blood. 129(4): 424–447). Unfortunately, relapse after conventional treatment is common due to chemotherapy resistance of leukemia stem cells (Eppert et al., 2011. Nat. Med. 17(9): 1086–1093), and current treatment options for relapsed / refractory (R / R) AML are poor, resulting in a 12-month overall survival rate of less than 30%.

[0016] Full-length CD33 protein (CD33 FL) is a transmembrane glycoprotein characterized by a distal V-set immunoglobulin (Ig)-like domain and a proximal C2-set Ig-like domain at the amino terminus of its extracellular portion (Figure 1).

[0017] CD33 FL It is primarily presented on mature cells of the myeloid lineage and on mature cells, initially expressed on pluripotent myeloid progenitor cells. It is not found outside the hematopoietic system and is thought not to be expressed on pluripotent hematopoietic stem cells. Consistent with its role as a myeloid differentiation antigen, CD33 FL CD33 is widely expressed on malignant cells in patients with myeloid neoplasms; for example, in AML, it is found in at least a subset of AML blast cells in almost all cases, and may be found in leukemia stem cells in some cases. Due to this expression pattern, CD33 FL CD33 has been widely used as an antigen for targeted therapy of AML (Walter et al., Blood 119(26):pp. 6198-6208, 2012; Cowan et al., Front. Biosci. (Landmark Ed) 18:pp. 1311-1334, 2013; Laszlo et al., Blood Ref. 28(4):pp. 143-153, 2014; and Walter, Expert Opin Investig Drugs 27(4):pp. 339-348, 2018). Although unconjugated monoclonal CD33 antibodies have been proven ineffective in clinical practice, several recent randomized trials using the CD33 antibody-drug conjugate (ADC) gemtuzumab ozogamicin (GO) have demonstrated improved survival rates in a subset of patients with AML, establishing the value of the antibody in this disease. FLHowever, it has been confirmed to be the first, and so far, only, therapeutic target for immunotherapy of AML (Laszlo et al., Blood Rev. 28(4): pp. 143-153, 2014; Godwin et al., Leukemia 31(9) 31(9): pp. 1855-1868, 2017). In parallel with the development of new, more effective CD33-targeted therapies (e.g., antibody-drug conjugates, radioactive immunoconjugates, bispecific antibodies, chimeric antigen receptor [CAR]-modified T cells) to overcome the limitations pointed out in GO, interest in CD33 as a drug target for other malignant and non-malignant disorders is growing. These efforts include targeting CD33 splice variants that are not recognized by GO, as well as targeting CD33+ tumor cells in other hematological malignancies, CD33+ myeloid-derived suppressor cells (MDSCs) in various diseases, and normal CD33+ microglia in Alzheimer's disease (Walter, Expert Opin Biol Ther. 2020, 20(9):955-958).

[0018] However, some patients lack exon 2 and CD33 ΔE2 It expresses a truncated splice variant of CD33, which is referred to as CD33. ΔE2 It has been identified at the mRNA level in normal hematopoietic cells and leukemia cells. Regarding the latter, CD33 ΔE2 The mRNA was identified in 29 of the 29 AML patient samples tested, demonstrating universal expression in human AML. CD33 ΔE2 This variant contains the C2 set Ig-like domain of CD33 but does not contain the V set Ig-like domain (Figure 1). Further splice variants identified at the mRNA level include CD33 E7a and CD33 ΔE2 / E7a CD33 is included. E7a This utilizes selective exon 7 (E7a) to induce truncation of the intracellular domain of CD33. ΔE2 / E7a It lacks exon 2 and also has truncation of the intracellular domain of CD33.

[0019] However, currently, almost all commercially available diagnostic CD33 antibodies and CD33 antibody-based therapies currently available clinically recognize the immunodominant V-set Ig-like domain encoded by exon 2 (Figure 1). That is, CD33 lacking the V-set Ig-like domain. ΔE2 And other CD33 proteins are not recognized by most commercially and clinically available CD33 antibodies. This means that these antibodies do not recognize CD33 ΔE2 This means that shorter forms of CD33, such as those lacking the V-set domain, will not be recognized. ΔE2 Selective transcription and CD33 FL This can explain the observation made in one clinical trial in pediatric AML that patients with a single nucleotide polymorphism in the CD33 gene, resulting in reduced translation, did not benefit from the addition of GO (which also binds to the V-set domain of CD33) to intensive chemotherapy.

[0020] Antibodies that recognize and bind to the C2 set Ig-like domain of the CD33 protein, regardless of the presence or absence of the V set Ig-like domain (e.g., CD33 PAN CD33, also known as an antibody ΔE2 and CD33 FLAntibodies that bind to isoforms represent a significant advance in targeting all CD33 isoforms, leading to broader therapeutic effects. These pan-binding antibodies are also expected to represent an advance because they bind proximal to the cell membrane (Figure 1). For several therapeutic targets, the specificity of the targeted epitope has been shown to be crucial for antibody-based therapies, and as shown for CD20, CD22, CD25, and EpCAM, proximal membrane epitopes provide more potent antitumor effects than distal membrane epitopes. For example, see Cleary et al., J Immunol. 2017; 198(10): pp. 3999-4011; Lin, Pharmgenomics Pers Med. 2010; 3: pp. 51-59; Haso et al., Blood. 2013; 121(7): pp. 1165-1174; and Bluemel et al., Cancer Immunol Immunother. 2010; 59(8): pp. 1197-1209.

[0021] This disclosure provides novel pan-binding antibodies that bind to the C2-set Ig-like domain of CD33, regardless of the presence or absence of the V-set Ig-like domain. PAN The conjugated antibodies include 1H10, 1A9, 1E6, 1D2, and 1B9.

[0022] This disclosure also provides newly developed anti-CD33 antibodies that bind to the V-set Ig-like domain of CD33. These V-set conjugates include 1H8, 2D3, and 2E3, and CD33 FL This provides additional diagnostic and treatment options for patients exhibiting the condition.

[0023] In certain embodiments, the antibodies disclosed herein bind to CD33 and have one or more of the following characteristics: (a) bind to recombinant human CD33; (b) bind to endogenous CD33 on the surface of human peripheral blood mononuclear cells (PBMCs) or other myeloid or non-myeloid human cells; (c) bind to endogenous CD33 on the surface of cancer cells; (d) bind to endogenous CD33 on the surface of AML cancer cells; (e) bind to an epitope in the CD33 C2 set Ig-like domain in the presence of a V set Ig-like domain (e.g., CD33 FL In the absence of a V-set Ig-like domain, CD33 binds to an epitope within the C2-set Ig-like domain (for example, CD33). ΔE2 (g) Binds to a proximal epitope on the membrane of CD33; (h) V of 1H10 L Chain and V H Including the chain; (i) V of 1A9 L Chain and V H Including the chain; (j) V of 1E6 L Chain and V H Including chain; (k)1D2 V L Chain and V H Including chains; (l) V of 1B9 L Chain and V H Including chain; (m)1H8 V L Chain and V H Includes chain; (n) 2D3 V L Chain and V H Including chain; (o) 2E3 V L Chain and V H The set includes a chain; (p) a CDR set of 1H10; (q) a CDR set of 1A9; (r) a CDR set of 1E6; (s) a CDR set of 1D2; (t) a CDR set of 1B9; (u) a CDR set of 1H8; (v) a CDR set of 2D3; and / or (w) a CDR set of 2E3. The CDR set can be any way predicted by IGMT, Kabat, North, Chothia, or "Set5".

[0024] The various forms of antibodies and their conjugated fragments described herein may be referred to herein as CD33 targeting agents.

[0025] While important aspects of this disclosure have been highlighted, further details and options for carrying out this disclosure are provided below: (i) CD33 antibodies; (ii) anti-CD33 antibody conjugates; (iii) anti-CD33 multispecific antibodies; (iv) formulations; (v) immune cell sampling and cell enrichment; (vi) genetic modification of cell populations to express recombinant proteins; (vii) cell activation culture conditions; (viii) ex vivo manufactured cell formulations; (ix) methods of use; (x) reference levels derived from control populations; (xi) exemplary embodiments; (xii) experimental examples; and (xiii) final paragraph. These headings are provided for compositional purposes only and do not limit the scope or interpretation of this disclosure.

[0026] (i) CD33 antibody. CD33 refers to any naturally occurring mature CD33 resulting from the processing of CD33 precursor protein within a cell. CD33-positive cells refer to any cells that express CD33 on their surface. CD33-positive cancers refer to cancers that contain one or more cells that express CD33 on their surface. Examples of CD33-positive cancers include leukemia, myelosarcoma, and lymphoma. More specific examples of such cancers include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute promyelocytic leukemia (APL), myeloproliferative neoplasms, megakaryocytic leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma (MM) and other plasma cell proliferation disorders, mast cell diseases, mast cell leukemia, mast cell sarcoma and myelosarcoma.

[0027] This disclosure provides antibodies that target the C2 set Ig-like domain of CD33 regardless of the presence or absence of the V set Ig-like domain, and antibodies that target the V set Ig-like domain of CD33. In certain embodiments, combinations of antibodies can be selected. For example, if the subject expresses the V set domain, a combination therapy including one or more of 1A9, 1H10, 1B9, 1E6, and 1D2 can be selected in combination with one or more of 2E3, 2D3, and 1H8. If the subject does not express the V set domain, 2E3, 2D3, and 1H8 are not administered.

[0028] Naturally occurring antibody structural units include tetramers. Each tetramer contains two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminus of each chain contains a variable region involved in antigen recognition and epitope binding. The variable region exhibits the same general structure of a relatively conserved framework region (FR) joined by three hypervariable regions, also called complementarity-determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework region, which enables binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain typically follows the Kabat sequences of proteins of immunological interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or the definitions of Chothia & Lesk, J.Mol.Biol., 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989.

[0029] A clear description of the CDR and identification of residues containing the antibody binding site can be achieved by elucidating the structure of the antibody and / or the structure of the antibody-epitope complex. In certain embodiments, this can be achieved by methods such as X-ray crystallography. Alternatively, the CDR may be determined by comparison with a known antibody (linear sequence) without relying on the elucidation of the crystal structure. To determine the residues involved in binding, the cocrystal structure of the Fab (antibody fragment) bound to the target may be optionally determined. Software programs such as ABodyBuilder may also be used.

[0030] The carboxyl-terminal regions of each chain define constant regions that can be involved in effector functions, particularly in the heavy chain (Fc). Examples of effector functions include C1q binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0031] Within the entire light and heavy chains, the variable and constant regions are joined by the "J" region of amino acids, and the heavy chain also contains the "D" region of amino acids. See, for example, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)).

[0032] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including IgM1 and IgM2. IgA is similarly subdivided into subclasses, including IgA1 and IgA2.

[0033] As shown, antibodies bind to epitopes on antigens. The term antigen refers to a molecule or part of a molecule to which an antibody can bind. An epitope is a region of the antigen to which the variable region of an antibody binds. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural properties and / or specific charge properties. If the antigen is a protein or peptide, the epitope contains specific amino acids within that protein or peptide that come into contact with the variable region of the antibody.

[0034] In certain embodiments, an epitope represents a binding site on CD33 to which the corresponding variable region of an antibody binds. The variable region binds to a linear epitope (e.g., an epitope containing a stretch of 5 to 12 consecutive amino acids) or to a three-dimensional structure formed by the spatial arrangement of several short stretches of a protein target. A three-dimensional epitope recognized by the variable region, for example, an epitope recognition site or paratope of an antibody or antibody fragment, can be considered a feature of the three-dimensional surface of the epitope molecule. These features fit precisely (into) the corresponding binding site of the variable region, thereby facilitating binding between the variable region and its target protein (more generally, the antigen). In certain embodiments, an epitope can be considered to have two levels: (i) a "covered patch" which can be considered as the shadow that the variable region of the antibody casts on the antigen to which it binds; and (ii) individual involved side chain and skeletal residues that facilitate binding. Binding then results from a combination of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0035] In certain embodiments, the epitope of the antibody of this disclosure (i.e., the epitope to which the antibody binds) is found within the C2 set Ig-like domain of CD33. The epitope provides a "pan-binding" site, which is also found in the CD33 molecule, which also contains a V set Ig-like domain (e.g., CD33 FL (if applicable) or does not include (for example, CD33) ΔE2This means that the antibody binds regardless of whether (i.e., if it is the case that...) (Figure 1). In certain embodiments, the epitope on the C2 set Ig-like domain is a membrane-proximal epitope. In certain embodiments, the membrane-proximal epitope is located within 115 residues of the transmembrane region; within 100 residues of the transmembrane region; within 75 residues of the transmembrane region; within 50 residues of the transmembrane region; within 25 residues of the transmembrane region; or within 15 residues of the transmembrane region. In certain embodiments, the epitope of the antibody of this disclosure is found within the V set Ig-like domain of CD33.

[0036] In certain embodiments, "to combine" means that the variable region is 10 -8 M or less, or 10 in a specific embodiment. -5 M~10 -13 M, in a particular embodiment, 10 -5 M~10 -10 M, in a particular embodiment, 10 -5 M~10 -7 M, in a particular embodiment, 10 -8 M~10 -13 M or, in a specific embodiment, 10 -9 M~10 -13 This means that it associates with its target epitope at the dissociation constant of M (Kd or KD). This term may be further used to indicate that the variable region does not bind to other biomolecules present (for example, this is 10 -4 M or more, or 10 in a specific embodiment. -4 It binds to other biomolecules with a dissociation constant (Kd) of M to 1M.

[0037] In certain embodiments, Kd may be characterized using BIAcore. For example, in certain embodiments, Kd may be measured using a surface plasmon resonance assay with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C, along with an immobilized antigen CM5 chip in 10 response units (RUs). Briefly, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) may be activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen may be diluted to 5 μg / ml (0.2 μM) in 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to obtain a coupling protein in 10 response units (RUs). After antigen injection, 1 M ethanolamine may be injected to block unreacted groups. For reaction rate measurement, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off The ratio k can be calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir coupled model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is given by the ratio k off / k on It can be calculated as follows. For example, see Chen et al., J.Mol.Biol.293:865-881, 1999. The on rate obtained by the above surface plasmon resonance assay is 10 6 M -1 s -1If it exceeds this, the on rate can be determined by using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-passing) at 25°C, pH 7.2, in the presence of increasing antigen concentrations, when measured with a spectrophotometer such as an Aviv Instruments spectrophotometer with stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0038] Unless otherwise specified, the term “antibody” includes its variants, derivatives, and fragments (in addition to antibodies having the two full-length heavy chains and two full-length light chains described above), examples of which are listed below. Furthermore, unless expressly excluded, antibodies may include monoclonal antibodies, human antibodies, bispecific antibodies, triplicate antibodies, quadruplicate antibodies, multispecific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, and fragments thereof. In certain embodiments, antibodies may include antibody oligomers or multiforms.

[0039] A monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for hypothetical variant antibodies that contain naturally occurring mutations or arise during the production of monoclonal antibody preparations, although such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which contain different antibodies directed to different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed to a single epitope on an antigen. Therefore, the modifier "monoclonal" characterizes the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.

[0040] A "human antibody" is an antibody that contains an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences.

[0041] The "Human Consensus Framework" is based on human immunoglobulin V L or V H This framework represents the most commonly occurring amino acid residues in the selection of framework sequences. Generally, it is used in human immunoglobulin V. L or V H Sequence selection is from a subgroup of variable domain sequences. The sequence subgroups may be those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication pp. 91-3242, Bethesda MD. (1991), vols. 1-3. In certain embodiments, V LRegarding this, the subgroup is the subgroup Kappa I, as described by Kabat et al. (above). In a particular embodiment, V H Regarding this, the subgroup is subgroup III, as described by Kabat et al. (above).

[0042] With respect to the antibodies provided herein, the following CDR sets are provided. A CDR set refers to three light chain CDRs and three heavy chain CDRs that work together to bind to CD33.

[0043] [Table 1] JPEG2026123131000002.jpg50153

[0044] [Table 2] JPEG2026123131000004.jpg104153

[0045] [Table 3] JPEG2026123131000006.jpg161153

[0046] [Table 4] JPEG2026123131000008.jpg211153 JPEG2026123131000009.jpg19153

[0047] [Table 5] JPEG2026123131000011.jpg74153

[0048] In a particular embodiment, the 1H10 antibody has the following sequence:

[0049] [ka] Variable light chains including, array:

[0050] [ka] It includes variable heavy chains.

[0051] In a particular embodiment, the 1A9 antibody has the sequence:

[0052] [ka] Variable light chains including, array:

[0053] [ka] It includes variable heavy chains.

[0054] In a particular embodiment, the 1E6 antibody has the following sequence:

[0055] [ka] Variable light chains including, array:

[0056] [ka] It includes variable heavy chains.

[0057] In a particular embodiment, the 1D2 antibody has the following sequence:

[0058] [ka] Variable light chains including, array:

[0059] [ka] It includes variable heavy chains.

[0060] In a particular embodiment, the 1B9 antibody has the following sequence:

[0061] [ka] Variable light chains including, array:

[0062] [ka] It includes variable heavy chains.

[0063] In a particular embodiment, CD33 Vセット The antibody contains 1H8. In certain embodiments, the 1H8 antibody has the following sequence:

[0064] [ka] Variable light chains including, array:

[0065] [ka] It includes variable heavy chains.

[0066] In a particular embodiment, CD33 Vセット The antibody contains 2D3. In certain embodiments, the 2D3 antibody has the sequence:

[0067] [Chemistry] a variable light chain containing, and sequence:

[0068] [Chemistry] a variable heavy chain containing.

[0069] In certain embodiments, the CD33 Vセット antibody contains 2E3. In certain embodiments, the 2E3 antibody has the sequence:

[0070] [Chemistry] a variable light chain containing, and sequence:

[0071] [Chemistry] a variable heavy chain containing.

[0072] The antibodies disclosed herein can be utilized to prepare various forms of related binding domain molecules. For example, certain embodiments can include binding fragments of the antibody, such as Fv, Fab, Fab’, F(ab’)2 and single-chain Fv fragments (scFv) or any biologically effective fragment of an immunoglobulin that specifically binds to an epitope described herein.

[0073] In certain embodiments, antibody fragments are used. An "antibody fragment" means a portion of a full or whole length antibody that retains the ability to bind to an epitope. Antibody fragments can be produced by a variety of techniques including proteolytic digestion of intact antibodies as well as production by recombinant host cells (e.g., mammalian suspension cell lines, E. coli or phage) as described herein. Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies and linear antibodies.

[0074] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin connected by a short linker peptide. An Fv fragment is the V L and V H domains of a single arm of an antibody, lacking the constant regions. The two domains of the Fv fragment, V L and V H are encoded by separate genes, but they can be joined by a synthetic linker that allows them to pair, for example, using recombinant methods, to become a single protein chain in which the V L and V H regions form a monovalent molecule (single-chain Fv (scFv)). For further information on Fv and scFv, see, for example, Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore (eds.), Springer-Verlag, New York (1994) 269-315; WO1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0075] The linker sequences used to connect the VL and VH of scFv are generally 5 to 35 amino acids in length. In certain embodiments, the VL-VH linker comprises 5 to 35, 10 to 30 amino acids or 15 to 25 amino acids. Variations in linker length may retain or enhance activity and result in excellent effectiveness in activity studies. The linker sequence of scFv is generally a Gly-Ser linker, which is described in more detail elsewhere in this specification.

[0076] Further examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only the heavy chain variable region. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0077] Fab fragments are V L 、V HThe Fab fragment is a monovalent antibody fragment containing the CL and CH1 domains. The F(ab')2 fragment is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in the hinge region. For a discussion of the Fab and F(ab')2 fragments with increased in vivo half-lives, see U.S. Patent No. 5,869,046. The diabody contains two potentially bivalent epitope-binding sites. See, for example, EP0404097;WO1993 / 01161; and Holliger et al., Proc. Natl. Acad. Sci. USA, 90: pp. 6444-6448, 1993. Dual affinity retargeting antibodies (DART®; based on the Diabody format but featuring a C-terminal disulfide crosslink for greater stability (Moore et al., Blood, 117:4542-51, 2011)) may also be used. Antibody fragments may also include isolated CDRs. For an overview of antibody fragments, see Hudson et al., Nat. Med., 9:129-134, 2003.

[0078] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications is described herein.

[0079] In certain embodiments, variants (including Fc variants) are modified from the reference sequence to provide an administration benefit. Exemplary administration benefits may include (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) altered binding affinity for protein complex formation, (4) altered binding affinity, (5) reduced immunogenicity, and / or (6) extended half-life. The following disclosure describes these modifications with respect to application to antibodies, but modifications may also be applicable to other specific anti-CD33 binding domain formats (e.g., scFv, bispecific antibodies) where applicable.

[0080] In certain embodiments, the antibody may be mutated to increase its affinity for the Fc receptor. An exemplary mutation that increases affinity for the Fc receptor is G236A / S239D / A330L / I332E(GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), pp. 6181-6186, 2012. In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region. In certain embodiments, modifications resulting in altered C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC) are made in the Fc region, as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642 and in Idusogie et al., J.Immunol. 164: pp. 4178-4184, 2000.

[0081] In certain embodiments, it may be desirable to produce a cysteine-modified antibody, e.g., "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located in an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is thus positioned in an accessible site of the antibody, which can then be used to conjugate the antibody with other molecules, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described below. In certain embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0082] Antibody variants are provided that have carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at position 297 (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence changes in the antibody. Such fucosylated variants may have improved ADCC function. For example, WO2000 / 61739;WO2001 / 29246;WO2002 / 031140;US2002 / 0164328;WO2003 / 085119;WO2003 / 084570;US2003 / 0115614;US2003 / 0157108;US2004 / 0093621;US2004 / 0110704;US2004 / 0132140;US See 2004 / 0110282;US2004 / 0109865;WO2005 / 035586;WO2005 / 035778;WO2005 / 053742;Okazaki et al., J.Mol.Biol.336:pp.1239-1249 (2004); and Yamane-Ohnuki et al., Biotech.Bioeng.87:p.614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: pp. 533-545, 1986, and knockout cell lines such as those containing the alpha-1,6-fucosyltransferase gene, FUT8, or knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: p. 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4): pp. 680-688, 2006; and WO2003 / 085107).

[0083] In certain embodiments, modified antibodies include those in which one or more amino acids are substituted with non-amino acid components, or in which amino acids are conjugated to a functional group, or in which functional groups are otherwise associated with amino acids. Modified amino acids may be, for example, glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, or amino acids conjugated to an organic derivatizer. Amino acids may be modified, for example, concurrently or post-translation during recombinant production (e.g., N-linked glycosylation in the NXS / T motif during expression in mammalian cells) or by synthetic means. Modified amino acids may be located within the sequence or at the end of the sequence. Modifications also include nitrite constructs.

[0084] In certain embodiments, the variant includes a glycosylation variant in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the reference sequence. In certain embodiments, the glycosylation variant includes more or fewer N-linked glycosylation sites than the reference sequence. The N-linked glycosylation sites are characterized by the sequence:Asn-X-Ser or Asn-X-Thr, where the amino acid residue represented as X can be any amino acid residue except proline. Substitutions of amino acid residues to construct this sequence provide a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions to delete this sequence remove an existing N-linked carbohydrate chain. Rearrangements of N-linked carbohydrate chains are also provided in which one or more N-linked glycosylation sites (e.g., naturally occurring ones) are deleted and one or more new N-linked sites are created. Further antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or substituted with a different amino acid (e.g., serine) compared to the reference sequence. These cysteine ​​variants can be useful when antibodies need to be refolded into a biologically active conformation, such as after the isolation of insoluble inclusions. These cysteine ​​variants generally have fewer cysteine ​​residues than the reference sequence and typically have an even number to minimize interactions arising from unpaired cysteines.

[0085] PEGylation is a process in which polyethylene glycol (PEG) polymer chains are covalently conjugated with other molecules, such as proteins. Several methods for PEGylating proteins have been reported in the literature. For example, N-hydroxysuccinimide (NHS)-PEG is used to PEGylate lysine residues and N-terminal free amine groups of proteins; PEG with an aldehyde group is used to PEGylate the amino terminus of proteins in the presence of a reducing agent; PEG with a maleimide functional group is used to selectively PEGylate free thiol groups of cysteine ​​residues of proteins; and site-specific PEGylation of acetyl-phenylalanine residues can be performed.

[0086] Covalent bonding of proteins with PEG has been proven to be a useful method for extending the half-life of proteins in vivo (Abuchowski, A. et al., Cancer Biochem. Biophys., 1984, 7:175-186; Hershfield, M. et al., N.Engl.J.Medicine, 1987, 316:589-596; and Meyers, F.J. et al., Clin.Pharmacol.Ther., 49:307-313, 1991). Binding PEG to proteins not only protects the molecules from enzymatic degradation but also reduces their clearance rate from the body. The size of the PEG attached to the protein significantly affects the protein's half-life. The ability of PEGylation to reduce clearance generally depends on the total molecular weight of the modified protein, rather than the number of PEG groups attached to the protein. Typically, larger PEG groups result in a longer in vivo half-life for the attached protein. Furthermore, PEGylation reduces protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788; p. 248, 1984), alters protein immunogenicity (Abuchowski et al., J. Biol. Chem. 252: p. 3582, 1977), and can even increase protein solubility, as described in PCT publication number WO92 / 16221.

[0087] Several sizes of PEG suitable for producing proteins with the desired circulating half-life are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver7.1). Various active PEGs are used, including PEG aldehydes such as mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and mPEG propionaldehyde.

[0088] In certain embodiments, an antibody may be fused to or coupled with an Fc polypeptide containing an amino acid modification that extends the in vivo half-life of the antibody containing the modified Fc polypeptide compared to the half-life of a similar antibody containing the same Fc polypeptide without amino acid modification. In certain embodiments, the amino acid modification of the Fc polypeptide may include M252Y, S254T, T256E, M428L and / or N434S, and may be used together, separately, or in any combination. For example, M428L / N434S is a pair of mutations that increases the half-life of an antibody in serum, as described by Zalevsky et al., Nature Biotechnology 28, pp. 157-159, 2010. Other potentially useful modifications are described in U.S. Patent No. 7,083,784, U.S. Patent No. 7,670,600, U.S. Patent Publication No. 2010 / 0234575, PCT / US2012 / 070146, and Zwolak, Scientific Reports 7:15521, 2017. In certain embodiments, any substitution at any of the following amino acid positions in the Fc polypeptide may be considered an Fc modification that extends the half-life: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each or a combination of these modifications may be used to extend the half-life of the bispecific antibodies described herein.

[0089] In certain embodiments, Fc modifications include huIgG4 ProAlaAla, huIgG2m4, and / or huIgG2sigma mutations.

[0090] In certain embodiments, the antibodies disclosed herein are formed using the Daedalus expression system described by Pechman et al. (Am J Physiol 294:R1234-R1239, 2008). The Daedalus system utilizes the inclusion of a minimally ubiquitous chromatin opening element in the transdermal vector to help reduce or prevent genomic silencing and maintain decigram-level expression stability. This system can avoid the cumbersome and time-consuming steps of other protein production methods by utilizing the secretion pathway of serum-free adapted human suspension cell lines such as 293 Freestyle. Using an optimized lentiviral vector, yields of 20–100 mg / l of well-folded, post-modified, endotoxin-free proteins up to 70 kDa can be obtained in conventional small-scale (100 ml) cultures. At these yields, most proteins can be purified using a single-size exclusion chromatography step that is directly suitable for use in structural, biophysical, or therapeutic applications. Bandaranayake et al., Nucleic Acids Res., 39(21) 2011. In some cases, chromatographic purification may not be necessary due to the purity of the preparations made by the methods described herein.

[0091] (ii) Anti-CD33 antibody conjugates. Anti-CD33 antibody conjugates include anti-CD33 immunotoxins, antibody-drug conjugates (ADCs), fluorescent conjugates, and radioisotope conjugates.

[0092] Anti-CD33 immunotoxins. In certain embodiments, antibodies may also be formed as immunotoxins. Anti-CD33 immunotoxins include anti-CD33 antibodies disclosed herein that are conjugated to one or more cytotoxins (e.g., protein toxins, bacterial, fungal, plant or animal enzymatically active toxins or fragments thereof). The toxin may be any active substance that is harmful to cells. Frequently used plant toxins are classified into two classes: (1) holotoxins (or class II ribosome inactivating proteins) such as lysine, abrin, mistletoxin, and modesine, and (2) hemitoxins (class I ribosome inactivating proteins) such as pokeweed antiviral protein (PAP), saporin, bryodin 1, bouganin, and geronin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2: pp. 313-325 (2001). Toxins can be obtained from virtually any source and may be synthetic or natural products.

[0093] An immunotoxin containing multiple (e.g., four) cytotoxins for each binding domain can be prepared by partially reducing the binding domain with an excess reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 30 minutes. Then, the buffer can be exchanged by eluting through SEPHADEX G-25 resin containing 1 mM DTPA (diethylenetriaminepentaacetic acid) in Dulbecco's phosphate-buffered saline (DPBS). The eluate can be further diluted with DPBS, and the thiol concentration of the binding domain can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, e.g., 5-fold, of the linker-cytotoxin conjugate can be added and left at 4 °C for 1 hour, and the conjugation reaction can be quenched by the addition of a substantial excess, e.g., 20-fold, of cysteine. The resulting immunotoxin mixture can be purified with SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugates and, if desired, desalted and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, e.g., through a 0.2 μm filter, and freeze-dried if storage is desired.

[0094] Antibody-drug conjugates (ADCs) enable the targeted delivery of the drug moiety to CD33-expressing cells and, in certain embodiments, allow for intracellular accumulation, where systemic administration of the unconjugated drug can result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0095] In certain embodiments, ADCs refer to targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic agents by targeting antigen-expressing cancer cells with potent cytotoxic agents (Teicher, BA (2009) Current Cancer Drug Targets 9: pp. 982-1004), thereby increasing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3): pp. 154-169; Chari, RV (2008) Acc. Chem. Res. 41: pp. 98-107). See also Kamath & Iyer (Pharm Res. 32(11): pp. 3470-3479, 2015) for considerations regarding the development of ADCs.

[0096] The drug portion (D) of the ADC may contain any compound, part, or group having cytotoxic or cytostatic effects. The drug portion may impart cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drugs include actinomycin D, anthracyclines, auristatin, calicheamicin, camptothecin, CC1065, colchicine, cytochalasin B, daunorubicin, 1-dehydrotestosterone, dihydroxyanthracinedione, dorastatin, doxorubicin, duocalmycin, erinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, and meitansinoids (monomethicin). This includes luaristatin E [MMAE] (containing vedotin), mitramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloids, vincristine, and their stereoisomers, homologs, analogs, and derivatives having cytotoxic activity.

[0097] Drugs can be obtained from virtually any source and may be synthetic products or natural products isolated from selected sources, such as plants, bacteria, insects, mammals, or fungal sources. Drugs may also be synthetically modified natural products or analogs of natural products.

[0098] The ADC compounds of this disclosure include those having anti-CD33 activity. In certain embodiments, the ADC compound includes an antibody conjugated, i.e., covalently bound to a drug moiety. In certain embodiments, the antibody is covalently bound to the drug moiety via a linker. The linker may include any chemical moiety that can link an antibody, antibody fragment (e.g., an antigen-binding fragment), or functional equivalent to another moiety, such as a drug moiety. The linker may be susceptible to cleavage, such as acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the compound or antibody remains active (cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or incleavable linker). In some embodiments, the linker is a procharge linker, a hydrophilic linker, or a dicarboxylic acid-based linker. ADCs selectively deliver an effective dose of drug to cancer cells, thereby achieving higher selectivity, i.e., a lower effective dose, while increasing the therapeutic index ("therapeutic window").

[0099] To prepare ADCs, linker-cytotoxin conjugates can be prepared by conventional methods similar to those described by Doronina et al. (Bioconjugate Chem. 17: pp. 114-124, 2006). Antibody-drug conjugates containing multiple (e.g., four) drugs per antibody can be prepared by partially reducing the antibody with an excess reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 minutes, after which the buffer can be replaced by elution through SEPHADEX G-25 resin containing 1 mM DTPA in Dulbecco's phosphate-buffered saline (DPBS). The eluate can be further diluted with DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ermann's reagent]. An excess, e.g., 5 times the linker-cytotoxin conjugate, can be added at 4°C and left for 1 hour. The conjugation reaction can be quenched by adding a considerably excess, e.g., 20 times the amount of cysteine. The resulting ADC mixture can be purified with SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, and if desired, it can be desalted and purified by size exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 μm filter, and if storage is desired, it can be lyophilized.

[0100] The anti-CD33 fluorescent conjugate comprises a CD33-binding domain linked to a fluorescent label. The fluorescent label may include any suitable label or a detectable group that can be detected by optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0101] Fluorescent labeling can be particularly useful in applications of cell staining, identification, and isolation. Exemplary fluorescent labels include: blue fluorescent proteins (e.g., eBFP, eBFP2, azurite, mKalama1, GFPuv, sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, emerald, azami green, monomeric azami green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green (Trademark) (Thermo Fisher). This includes luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red (trademark) (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0102] Anti-CD33 radioisotope conjugates contain a CD33-binding domain linked to a radioisotope for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / or treatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to subjects. Therapeutic nuclear medicine is often referred to as radiotherapy or radioimmunotherapy (RIT).

[0103] Examples of radioisotopes that can be conjugated to the antibodies of the present disclosure include iodine-131, arsenic-72, arsenic-74, iodine-131, indium-111, yttrium-90, and lutetium-177, as well as alpha-emitting radionuclides such as astatine 211, actinium-225, bismuth-212, or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates, including Zevalin (trademark) (DEC Pharmaceuticals), are commercially available, and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the present disclosure.

[0104] Examples of radionuclides useful in radiotherapy include 225 Ac and 227 Th. 225 Ac is a radionuclide with a half-life of 10 days. 225 When Ac decays, the daughter isotope 221 Fr, 213 Bi, and 209 Pb are formed. 227 Th has a half-life of 19 days and forms the daughter isotope 223 Ra.

[0105] Further examples of useful radioisotopes include 228 Ac, 111 Ag, 124 Am, 74 As, 211 At, 209 At, 194 Au, 128 Ba, 7 Be, 206 Bi, 245 Bk, 246 Bk, 76 Br, 11 C, 47 Ca, 254 Cf, 242 Cm, 51 Cr, 67 Cu, 153 Dy, 157 Dy, 159 Dy, 165 Dy, 166 Dy,171 Do, 250 Es、 254 Es、 147 Eu、 157 Eu、 52 Fe、 59 Fe、 251 Fm、 252 Fm、 253 Fm、 66 Won't, 72 Won't, 146 Gd、 153 Gd、 68 Here、 170 Hf、 171 Hf、 193 Hg、 193 mHg、 160 mHo、 130 I、 131 I、 135 I、 114 mIn、 185 They、 42 K、 43 K、 76 Kr、 79 Kr、 81 mKr、 132 That, 262 Lr、 169 Is, 174 I was, 176 I was, 257 Md、 260 Md、 28 Mg、 52 Mn、 90 I, 24 Provide, 95 Nb、 138 Nd、 57 Ni、 66 Ni、 234 Np、 15 O、 182 Os、 189 mOs、 191 Os、 32 P、 201 Pb、 101 Pd、 143 Pr、 191 Pt、 243 Pu、 225 Ra、 81 Rb、 188 Re、 105 Rh、 211 Rn、 103 Ru、 35S, 44 Sc, 72 Se, 153 Sm, 125 Sn, 91 Sr, 173 Ta, 154 Tb, 127 Te, 234 Th, 45 Ti, 166 Tm, 230 U, 237 U, 240 U, 48 V, 178 W, 181 W, 188 W, 125 Xe, 127 Xe, 133 Xe, 133 mXe, 135 Xe, 85 mY, 86 Y, 90 Y, 93 Y, 169 Yb, 175 Yb, 65 Zn, 71 mZn, 86 Zr, 95 Zr and / or 97 Zr is included.

[0106] (iii) Anti-CD33 multispecific antibodies. Anti-CD33 bispecific antibodies have at least one epitope that binds to at least two epitopes located on CD33. Anti-CD33 tripspecific antibodies have at least one epitope that binds to at least three epitopes located on CD33, and so on.

[0107] Bispecific antibodies may be prepared as full-length antibodies or as antibody fragments (e.g., F(ab')2 bispecific antibodies). For example, WO1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody, U.S. Patent No. 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma RI antibody, WO1998 / 002463 describes a bispecific anti-ErbB2 / Fc alpha antibody, and U.S. Patent No. 5,821,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody. In certain embodiments, the bispecific antibody may be in the form of a bispecific T cell induction (BiTE®) antibody.

[0108] Some further exemplary bispecific antibodies have two heavy chains (each with three heavy chain CDRs followed by a CH1 domain, hinge, CH2 domain, and CH3 domain (N-terminus to C-terminus)) and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as shown, further configurations are envisioned that include bispecific antibodies in which the light chains associate with each heavy chain but do not contribute (or contribute minimally) to antigen-binding specificity, or can bind to one or more epitopes to which the heavy chain antigen-binding region binds, or can associate with each heavy chain and enable the binding of one or both heavy chains to one or both epitopes.

[0109] Instead of the whole antibody, scFv dimers or diabodies may be used. Diabodies and scFvs can be constructed using only the variable domain (usually containing variable domain components from both the light and heavy chains of the source antibody) without the Fc region, which may reduce the influence of anti-idiotype reactions. Other forms of bispecific antibodies include single-stranded "Janusins" described by Traunecker et al. (Embo Journal, 10, pp. 3655-3659, 1991).

[0110] Bispecific antibodies with extended half-lives are described, for example, in U.S. Patent No. 8,921,528 and U.S. Patent Publication No. 2014 / 0308285.

[0111] Methods for producing bispecific antibodies are known in the art. For example, conventional production of full-length bispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy-light chain pairs, each having two distinct chains (see, e.g., Millstein et al., Nature 305: pp. 37-39, 1983). Similar procedures are disclosed, for example, in WO1993 / 008829, Traunecker et al., EMBO J.10: pp. 3655-3659, 1991, and Holliger & Winter, Current Opinion Biotechnol.4, pp. 446-449 (1993).

[0112] In certain embodiments, bispecific antibodies can be prepared using chemical bonding. For example, Brennan et al. (Science 229: p. 81, 1985) describe a procedure in which an intact antibody is proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody.

[0113] In certain embodiments, the binding domains disclosed herein can be used to construct bispecific, tripspecific, (or more specific) immune cell-inducing antibody constructs.

[0114] In certain embodiments, the binding domains disclosed herein can be used to construct bispecific, tripspecific, (or more specific) immune cell-inducing antibody constructs. These immune cell-inducing antibody constructs can induce, for example, T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or mixtures of HSCs and HPCs (i.e., HSPCs). In certain embodiments, the immune cell-inducing antibody construct induces T cells.

[0115] Several different subsets of T cells have been discovered, each with a different function. For example, most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called the α-TCR chain and the β-TCR chain.

[0116] γδ T cells represent a small subset of T cells that possess different T cell receptors (TCRs) on their surface. In γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is less common than αβ T cells (2% of all T cells).

[0117] CD3 is expressed on all mature T cells. Activated T cells express 4-1BB (CD137), CD69, and CD25. CD5 and the transferrin receptor are also expressed on T cells.

[0118] T cells can be further classified into helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. T helper cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that modulate or assist the active immune response.

[0119] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in graft rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost all cells in the body.

[0120] As used herein, “central memory” T cells (or “TCM”) refer to CTLs that have experienced an antigen and express CD62L or CCR7 and CD45RO on their surface, and do not express or have reduced expression of CD45RA compared to naive cells. In certain embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO and CD95, and have reduced expression of CD45RA compared to naive cells.

[0121] As used herein, “effector memory” T cells (or “TEM”) refer to T cells that have experienced an antigen and which do not express or have reduced expression of CD62L on their surface compared to central memory cells, and do not express or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for CD62L and CCR7 expression and have variability in CD28 and CD45RA expression compared to naive or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0122] As used herein, “naive” T cells refer to T cells that have not experienced an antigen and, compared to central memory cells or effector memory cells, express CD62L and CD45RA but not CD45RO. In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers including CD62L, CCR7, CD28, CD127, and CD45RA.

[0123] Natural killer cells (NK cells, K cells, and killer cells) are activated in response to interferon or macrophage-derived cytokines. They function to suppress viral infections while antigen-specific cytotoxic T cells, which can eliminate infections through an adaptive immune response, are being generated. NK cells express CD8, CD16, and CD56, but do not express CD3.

[0124] NK cells include NK-T cells. NK-T cells are a specialized population of T cells that express a semi-invariant T cell receptor (TCR ab) and surface antigens that typically associate with natural killer cells. NK-T cells contribute to antimicrobial and antiviral immune responses and promote tumor-related immune surveillance or immunosuppression. Like natural killer cells, NK-T cells can also induce perforin-related, Fas-related, and TNF-related cytotoxicity. Activated NK-T cells can produce IFN-γ and IL-4. In certain embodiments, NK-T cells are CD3+ / CD56+.

[0125] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body (in certain cases, as microglia, Kupffer cells, and osteoclasts) and engulf apoptotic cells, pathogens, and other non-self components. Monocytes / macrophages express CD11b, F4 / 80, CD68, CD11c, IL-4Rα, and / or CD163.

[0126] Immature dendritic cells (i.e., pre-activation) engulf antigens and other non-self components in the periphery, and then, in their activated form, migrate to the T cell region of lymphoid tissue to present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0127] Hematopoietic stem cells / progenitor cells, or HSPCs, refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0128] Hematopoietic stem cells are undifferentiated hematopoietic cells that can self-replicate in vivo, proliferate essentially without limit in vitro, and differentiate into all other hematopoietic cell types.

[0129] Hematopoietic progenitor cells are cells derived from hematopoietic stem cells or fetal tissue that can further differentiate into mature cell types. In certain embodiments, hematopoietic progenitor cells are CD24 lo Lin - CD117+ Hematopoietic progenitor cells. HPCs can differentiate into (i) myeloid progenitor cells that ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) lymphoid progenitor cells that ultimately give rise to T cells, B cells and NK cells.

[0130] HSPCs may be positive for certain markers expressed at increased levels on HSPCs compared to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or combinations thereof. Conversely, HSPCs may be negative for expression markers compared to other types of hematopoietic cells. For example, such markers include Lin, CD38, or combinations thereof. Preferably, HSPCs are CD34 + It is a cell.

[0131] The statement that a cell or cell population is “positive” for a particular marker or expresses a particular marker refers to the detectable presence of the particular marker on or within the cell. When referring to a surface marker, the term may refer to the presence of surface expression that can be detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the stain is detectable by flow cytometry at a level substantially higher than the stain detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that for cells known to be positive for the marker, and / or at a level substantially higher than that for cells known to be negative for the marker.

[0132] The statement that a cell or cell population is “negative” for a particular marker or lacks expression of the marker means that there is substantially no detectable presence of the particular marker on or within the cell. When referring to a surface marker, this term may refer to the absence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the stain is not detected by flow cytometry at a level substantially higher than the stain detected by the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than the level for cells known to be positive for the marker, and / or at a level substantially similar to the level for cells known to be negative for the marker.

[0133] Examples of multispecific immune cell-inducing antibody constructs include those that bind to both CD33 and immune cell (e.g., T cells or NK cells) activating epitopes, with the aim of attracting immune cells to CD33-expressing cells and destroying them. See, for example, US2008 / 0145362. Such constructs are referred to herein as immunoactivating multispecific (I-AMS). BiTEs® (Amgen, Thousand Oaks, CA) is one form of I-AMS. Immune cells that may be targeted for local activation by I-AMS in this disclosure include, for example, T cells, natural killer (NK) cells, and macrophages, which are discussed in more detail herein.

[0134] T cell activation can be mediated by two distinct signals: a signal that initiates antigen-dependent primary activation and provides a T cell receptor-like signal (primary cytoplasmic signaling sequence) and a signal that acts antigen-independently and provides a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequence). The I-AMS disclosed herein can target any T cell activation epitope that induces T cell activation upon binding. Examples of such T cell activation epitopes are found on T cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB (CD137), OX40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3.

[0135] In a particular embodiment, the CD3 binding domain is in the sequence:

[0136] [ka] Variable light chain (LcFv) and sequence including:

[0137] [ka] It contains variable heavy chains (HcFv).

[0138] In certain embodiments, the CD3 binding domain (e.g., scFv) is derived from an OKT3 antibody (the same one used in blinatumomab). The OKT3 antibody is described in detail in U.S. Patent No. 5,929,212. It comprises a variable light chain comprising a CDRL1 sequence containing SASSSVSYMN (SEQ ID NO: 67), a CDRL2 sequence containing RWIYDTSKLAS (SEQ ID NO: 68), and a CDRL3 sequence containing QQWSSNPFT (SEQ ID NO: 69). In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain comprising a CDRH1 sequence containing KASGYTFTRYTMH (SEQ ID NO: 70), a CDRH2 sequence containing INPSRGYTNYNQKFKD (SEQ ID NO: 71), and a CDRH3 sequence containing YYDDHYCLDY (SEQ ID NO: 72).

[0139] The following sequence is an scFv derived from OKT3 that retains the ability to bind to CD3:

[0140] [ka] This can also be used as a CD3 binding domain.

[0141] In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing QSLVHNNGNTY (SEQ ID NO: 74), a CDRL2 sequence containing KVS, and a CDRL3 sequence containing GQGTQYPFT (SEQ ID NO: 75). In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTFTKAW (SEQ ID NO: 76), a CDRH2 sequence containing IKDKSNSYAT (SEQ ID NO: 77), and a CDRH3 sequence containing RGVYYALSPFDY (SEQ ID NO: 78). These reflect the CDR sequences of the 20G6-F3 antibody.

[0142] In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing QSLVHDNGNTY (SEQ ID NO: 79), a CDRL2 sequence containing KVS, and a CDRL3 sequence containing GQGTQYPFT (SEQ ID NO: 75). In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTFSNAW (SEQ ID NO: 80), a CDRH2 sequence containing IKARSNNYAT (SEQ ID NO: 81), and a CDRH3 sequence containing RGTYYASKPFDY (SEQ ID NO: 82). These reflect the CDR sequences of the 4B4-D7 antibody.

[0143] In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing QSLEHNNGNTY (SEQ ID NO: 83), a CDRL2 sequence containing KVS (not included in the sequence listing), and a CDRL3 sequence containing GQGTQYPFT (SEQ ID NO: 75). In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTFSNAW (SEQ ID NO: 80), a CDRH2 sequence containing IKDKSNNYAT (SEQ ID NO: 84), and a CDRH3 sequence containing RYVHYGIGYAMDA (SEQ ID NO: 85). These reflect the CDR sequences of the 4E7-C9 antibody.

[0144] In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing QSLVHTNGNTY (SEQ ID NO: 86), a CDRL2 sequence containing KVS, and a CDRL3 sequence containing GQGTHYPFT (SEQ ID NO: 87). In certain embodiments, the CD3 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTFTNAW (SEQ ID NO: 88), a CDRH2 sequence containing KDKSNNYAT (SEQ ID NO: 89), and a CDRH3 sequence containing RYVHYRFAYALDA (SEQ ID NO: 90). These reflect the CDR sequences of the 18F5-H10 antibody.

[0145] Further examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WO2016 / 116626. TR66 may also be used.

[0146] CD28 is a surface glycoprotein present in 80% of human peripheral T cells, and is found in both resting and active T cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent known co-stimulatory molecule (June et al., Immunol. Today 15: p. 321, 1994; Linsley et al., Ann. Rev. Immunol. 11: p. 191, 1993). In certain embodiments, the CD28-binding domain (e.g., scFv) is derived from CD80, CD86, or 9D7 antibodies. Further antibodies that bind to CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10. Furthermore, 1YJD provides a crystal structure of human CD28 complexed with the Fab fragment of a mitotic antibody (5.11A1).

[0147] In certain embodiments, the CD28 binding domain is derived from TGN1412. In certain embodiments, the variable heavy chain of TGN1412 is

[0148] [ka] The variable light chain of TGN1412 includes,

[0149] [ka] Includes.

[0150] In certain embodiments, the CD28-binding domain includes a variable light chain comprising a CDRL1 sequence containing HASQNIYVWLN (SEQ ID NO: 93), a CDRL2 sequence containing KASNLHT (SEQ ID NO: 94), and a CDRL3 sequence containing QQGQTYPYT (SEQ ID NO: 95), and a variable heavy chain comprising a CDRH1 sequence containing GYTFTSYYIH (SEQ ID NO: 96), a CDRH2 sequence containing CIYPGNVNTNYNEK (SEQ ID NO: 97), and a CDRH3 sequence containing SHYGLDWNFDV (SEQ ID NO: 98).

[0151] In certain embodiments, the CD28 binding domain includes a variable light chain comprising a CDRL1 sequence containing HASQNIYVWLN (SEQ ID NO: 93), a CDRL2 sequence containing KASNLHT (SEQ ID NO: 94), and a CDRL3 sequence containing QQGQTYPYT (SEQ ID NO: 95), as well as a variable heavy chain comprising a CDRH1 sequence containing SYYIH (SEQ ID NO: 99), a CDRH2 sequence containing CIYPGNVNTNYNEKFKD (SEQ ID NO: 100), and a CDRH3 sequence containing SHYGLDWNFDV (SEQ ID NO: 98).

[0152] Activated T cells express 4-1BB(CD137). In certain embodiments, the 4-1BB binding domain includes a variable light chain containing a CDRL1 sequence including RASQSVS (SEQ ID NO: 101), a CDRL2 sequence including ASNRAT (SEQ ID NO: 102), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 103), as well as a variable heavy chain containing a CDRH1 sequence including YYWS (SEQ ID NO: 104), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 105).

[0153] In certain embodiments, the 4-1BB binding domain includes a variable light chain comprising a CDRL1 sequence containing SGDNIGDQYAH (SEQ ID NO: 106), a CDRL2 sequence containing QDKNRPS (SEQ ID NO: 107), and a CDRL3 sequence containing ATYTGFGSLAV (SEQ ID NO: 108), as well as a variable heavy chain comprising a CDRH1 sequence containing GYSFSTYWIS (SEQ ID NO: 109), a CDRH2 sequence containing KIYPGDSYTNYSPS (SEQ ID NO: 110), and a CDRH3 sequence containing GYGIFDY (SEQ ID NO: 111).

[0154] Certain embodiments disclosed herein include a binding domain that binds to an epitope on CD8. In certain embodiments, the CD8 binding domain (e.g., scFv) is derived from an OKT8 antibody. For example, in certain embodiments, the CD8 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing RTSRSISQYLA (SEQ ID NO: 112), a CDRL2 sequence containing SGSTLQS (SEQ ID NO: 113), and a CDRL3 sequence containing QQHNENPLT (SEQ ID NO: 114). In certain embodiments, the CD8 T cell activation epitope binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFNIKD (SEQ ID NO: 115), a CDRH2 sequence containing RIDPANDNT (SEQ ID NO: 116), and a CDRH3 sequence containing GYGYYVFDH (SEQ ID NO: 117). These reflect the CDR sequences of the OKT8 antibody.

[0155] In certain embodiments, natural killer cells (also known as NK cells, K cells, and killer cells) are targeted for local activation by I-AMS. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt the cell membrane and can secrete cytokines to recruit other immune cells.

[0156] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, and several members of the innate cytotoxic receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0157] Examples of commercially available antibodies that bind to NK cell receptors and induce and / or enhance NK cell activation include 5C6 and 1D11 (available from BioLegend® San Diego, CA) which bind to and activate NKG2D; mAb33 (available from BioLegend®) which binds to and activates KIR2DL4; P44-8 (available from BioLegend®) which binds to and activates NKp44; SK1 which binds to and activates CD8; and 3G8 which binds to and activates CD16.

[0158] In certain embodiments, I-AMS can bind to and block NK cell inhibitory receptors to enhance NK cell activation. Examples of NK cell inhibitory receptors that can be bound to and blocked include KIR2DL1, KIR2DL2 / 3, KIR3DL1, NKG2A, and KLRG1. In certain embodiments, the binding domain that binds to and blocks the NK cell inhibitory receptors KIR2DL1 and KIR2DL2 / 3 is sequence

[0159] [ka] Variable light chain region and arrangement

[0160] [ka] It includes a variable heavy chain region. Further NK cell activating antibodies are described in WO / 2005 / 0003172 and U.S. Patent No. 9,415,104.

[0161] In certain embodiments, macrophages are targeted for local activation by I-AMS. Macrophages are a type of white blood cell (or leukocyte) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis.

[0162] I-AMS can be designed to bind to proteins expressed on the surface of macrophages. Examples of activated proteins expressed on the surface of macrophages (and their precursor cells, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Rα, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70 (available from BioLegend®) which binds to and activates CD11b; KP1 (available from ABCAM®, Cambridge, United Kingdom) which binds to and activates CD68; and ab87099 (available from ABCAM®) which binds to and activates CD163.

[0163] In certain embodiments, I-AMS can target pathogen recognition receptors (PRRs). PRRs are proteins or protein complexes that recognize danger signals and activate and / or enhance innate immune responses. Examples of PRRs include the TLR4 / MD-2 complex that recognizes Gram-negative bacteria; Dectin-1 and Dectin-2 that recognize mannose moieties on fungi and other pathogens; the TLR2 / TLR6 or TLR2 / TLR1 heterodimer that recognizes Gram-positive bacteria; TLR5 that recognizes flagellin; and TLR9 (CD289) that recognizes CpG motifs in DNA. In certain embodiments, I-AMS can bind to and activate TLR4 / MD-2, Dectin-1, Dectin-2, TLR2 / TLR6, TLR2 / TLR1, TLR5 and / or TLR9.

[0164] In certain embodiments, I-AMS can target the complement system, which is an immune pathway induced by antigen-binding antibodies and involving complement protein signaling, resulting in the immune recognition and clearance of antibody-coated antigens.

[0165] The binding domains of I-AMS and other manipulated formats described herein may be joined via a linker. The linker is an amino acid sequence that can provide flexibility and room for conformational shifts between the I-AM binding domains. Any suitable linker may be used.

[0166] An example of a linker can be found in Chen et al., Adv Drug Deliv Rev. 2013 / 01 / 15;65(10):1357-1369. The linker can be flexible, rigid, or semi-rigid depending on the presentation of the desired functional domain to the target.

[0167] Commonly used flexible linkers include a linker sequence containing the amino acids glycine and serine (Gly-Ser linker). In certain embodiments, the linker sequence is (Gly x Ser y ) nIncludes sets of glycine and serine repeats such as (SEQ ID NO: 120) with 1 to 10 repeats, where x and y are independently integers from 0 to 10, except that both x and y are not 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A specific example is (Gly4Ser) n (Sequence ID 121), (Gly3Ser) n (Gly4Ser) n (Sequence ID 122), (Gly3Ser) n (Gly2Ser) n (Sequence ID 123) and (Gly3Ser) n (Gly4Ser)1 (SEQ ID NO: 124) is included. In certain embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 125), (Gly4Ser)3 (SEQ ID NO: 126), (Gly4Ser)2 (SEQ ID NO: 127), (Gly4Ser)1 (SEQ ID NO: 128), (Gly3Ser)2 (SEQ ID NO: 129), (Gly3Ser)1 (SEQ ID NO: 130), (Gly2Ser)2 (SEQ ID NO: 131) or (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 132), GGSGGGSGSG (SEQ ID NO: 133) or GGSGGGSG (SEQ ID NO: 134).

[0168] Linkers containing one or more antibody hinge regions and / or constant immunoglobulin heavy chain regions, such as CH3 alone or a CH2CH3 sequence, may also be used.

[0169] In some situations, flexible linkers may not be able to maintain the distance or arrangement of binding domains required for a particular application. In these examples, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In certain embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be predicted based on chance alone. In certain embodiments, a proline-rich linker is a linker having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. A specific example of a proline-rich linker is a fragment of proline-rich salivary protein (PRP).

[0170] The cytolytic properties of I-AMS molecules can be confirmed in comparative in vitro assays. Briefly, in cell line experiments, target cancer cells can be incubated in 96-well round-bottom plates with 5-10,000 cells / well containing increased concentrations of various I-AMS antibodies (e.g., CD33 / CD3 I-AMS containing CD33-CD3 bispecific antibody (BsAb)) with or without healthy donor T cells (used in 1:1 and 3:1 E:T cell ratios). After 48 hours, cell number and drug-induced cytotoxicity can be determined by flow cytometry using 4',6-diamidino-2-phenylindole (DAPI) to detect non-viable cells. In experiments where healthy donor T cells are added, cancer cells can be identified by forward / lateral scattering properties and negativity to CellVue Burgundy dye. Experiments may involve technical overlap.

[0171] In certain embodiments including the I-AMS construct, the T cell activation epitope binding domain is a known TCR. α , V β , C α Or C βCompared to the above, this includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or combinations of the above changes. Insertions, deletions, or substitutions include the amino terminus, carboxyl terminus, or both of these regions. α , V β , C α or C β Any location in the region is acceptable, provided that each CDR contains no changes, or contains one, two, or three or fewer changes, and is modified V α , V β , C α or C β The binding domain, including the region, can still specifically bind to its target with the same affinity as the wild type.

[0172] In certain embodiments, a bispecific molecule can be assembled by synthesizing each scFv as a DNA fragment having overlapping Gibson assembly compatible ends in a standard BiTE® antibody format, using a variable region CD33 antibody sequence derived from 5'RACE (rapid cDNA end cloning) cloning and a CD3 sequence from CD33-CD3 BsAb. A prototypical intervening region, such as the (Gly4Ser)3 (SEQ ID NO: 126) linker, can be used between the paired variable domains and a short Gly4Ser (SEQ ID NO: 128) linker between the two scFvs.

[0173] Anti-CD33 trispecific antibodies are artificial proteins that simultaneously bind to three different types of antigens, each having at least one CD33 antigen. Trispecific antibodies are described, for example, in WO2016 / 105450, WO2010 / 028796, WO2009 / 007124, WO2002 / 083738, US2002 / 0051780, and WO2000 / 018806.

[0174] (iv) Formulations. Any of the antibodies described herein in any exemplary format may be formulated into a composition, either alone or in combination, for administration to a subject. Salts and / or prodrugs of antibodies may also be used.

[0175] Pharmacopoeially acceptable salts include any salt that retains antibody activity and is pharmaceutically acceptable. Pharmacopoeially acceptable salts also refer to any salt that may be formed in vivo as a result of administration of an acid, another salt, or a prodrug converted to an acid or salt.

[0176] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic acid, and sulfonic acid classes.

[0177] Suitable pharmaceutically acceptable base addition salts include metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine, and procaine.

[0178] Prodrugs contain an active ingredient that, after administration, is converted into a therapeutically active compound, for example, by cleavage or hydrolysis of a biologically unstable group.

[0179] In a particular configuration, the composition contains antibodies in an amount of at least 0.1% w / v or w / w of the composition, at least 1% w / v or w / w of the composition, at least 10% w / v or w / w of the composition, at least 20% w / v or w / w of the composition, at least 30% w / v or w / w of the composition, at least 40% w / v or w / w of the composition, at least 50% w / v or w / w of the composition, at least 60% w / v or w / w of the composition, at least 70% w / v or w / w of the composition, at least 80% w / v or w / w of the composition, at least 90% w / v or w / w of the composition, at least 95% w / v or w / w of the composition, or at least 99% w / v or w / w of the composition.

[0180] Exemplary commonly used pharmaceutically acceptable carriers include any absorption retarder, antioxidant, binder, buffer, filler or bulking agent, chelating agent, coating, disintegrant, dispersion medium, gel, isotonic agent, lubricant, preservative, salt, solvent or co-solvent, stabilizer, surfactant and / or transport vehicle.

[0181] Examples of antioxidants include ascorbic acid, methionine, and vitamin E.

[0182] Exemplary buffers include citrate buffer, succinate buffer, tartarate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer, and / or trimethylamine salts.

[0183] An example of a chelating agent is EDTA (ethylenediaminetetraacetic acid).

[0184] Examples of isotonic agents include polyhydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0185] Exemplary preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0186] Stabilizers refer to a broad range of excipients, ranging in function from volume extenders to additives that help solubilize antibodies or prevent their denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols; amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, and cyclitol, e.g., inositol; PEG; amino acid polymers; sulfur-containing reducing agents, e.g., urea, glutathione. These may include: thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., less than 10 residues); proteins, e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; monosaccharides, e.g., xylose, mannose, fructose, and glucose; disaccharides, e.g., lactose, maltose, and sucrose; trisaccharides, e.g., raffinose; and polysaccharides, e.g., dextran. Stabilizers are typically present in the range of 0.1 to 10,000 parts by weight based on the weight of the therapeutic agent.

[0187] The compositions disclosed herein may be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein may further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intrafocal, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, oral, sublingual, and / or subcutaneous administration.

[0188] For injection, the composition may be formulated as an aqueous solution in a buffer containing, for example, Hanks' solution, Ringer's solution, or physiological saline. The aqueous solution may contain formulation agents, such as suspending agents, stabilizers, and / or dispersants. Alternatively, the formulation may be in lyophilized and / or powder form for preparation before use in a suitable vehicle, such as pyrogen-free sterile water.

[0189] For oral administration, the composition may be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. For oral solid dosage forms, such as powders, capsules, and tablets, suitable excipients include binders (tragacanth gum, gum arabic, corn starch, gelatin), fillers, such as sugars, such as lactose, sucrose, mannitol, and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binders. If desired, disintegrants such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, may be added. If desired, the solid dosage form may be sugar-coated or enterically coated using standard techniques. Flavorings such as peppermint, wintergreen oil, cherry flavor, and orange flavor may also be used.

[0190] The composition may be formulated as an aerosol. In certain embodiments, the aerosol is provided as part of an anhydrous, liquid, or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers may also be used with a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dosage unit may be determined by providing a valve for delivering a fixed amount. Gelatin capsules and cartridges for use in inhalers or blowers may also be formulated, containing the composition and a powder mixture with a suitable powder base, such as lactose or starch.

[0191] The composition may also be formulated as a depot preparation. The depot preparation may be formulated using a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, such as a sparingly soluble salt.

[0192] Furthermore, the composition may be formulated as a sustained-release system utilizing a semipermeable matrix of a solid polymer containing at least one type of antibody. Various sustained-release materials have been established and are well known to those skilled in the art. Depending on their chemical properties, the sustained-release system may release one or more antibodies over a period of several weeks to more than 100 days after administration. Depot preparations may be administered by injection; parenteral injection; ophthalmic instillation; or implantation into soft tissue, body cavities, or sometimes intravascularly via injection through a fine needle.

[0193] Depot formulations may contain various biodegradable polymers, including poly(lactide), poly(glycolide), poly(caprolactone), and poly(lactide)-co(glycolide) (PLG), with desired lactide:glycolide ratios, average molecular weights, polydispersity, and terminal group chemistry. By mixing different polymer types in different ratios using various grades, properties incorporated from each contributing polymer can be obtained.

[0194] The use of different solvents (e.g., dichloromethane, chloroform, ethyl acetate, triacetin, N-methylpyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter the size and structure of the particulate matter and modulate its release properties. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0195] Exemplary release modifiers may include surfactants, detergents, internal phase thickeners, complexing agents, surfactant molecules, cosolvents, chelating agents, stabilizers, cellulose derivatives, (hydroxypropyl)methylcellulose (HPMC), HPMC acetate, cellulose acetate, Pluronic® (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0196] Excipients distributed to the external phase boundaries of fine particles, such as surfactants containing polysorbate, dioctyl sulfosuccinate, poloxamer, and PVA, can also modify properties including particle stability and erosion rate, hydration and channel structure, interfacial transport, and reaction rate in a favorable manner.

[0197] Further processing of the disclosed sustained-release depot formulations may involve using stabilizing excipients, including mannitol, sucrose, trehalose, and glycine, in a buffer such as tris, citrate, or histidine, along with other components such as polysorbate, PVA, and dioctyl sulfosuccinate. A lyophilization cycle may also be used to produce a very low-moisture powder that is reconstituted to similar size and performance characteristics to the original suspension.

[0198] Any composition disclosed herein may favorably include any other pharmaceutically acceptable carrier, including any that does not cause significantly adverse allergic or other troublesome reactions that outweigh the benefits of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990. Furthermore, formulations may be prepared to meet the sterility, pyrogenicity, general safety, and purity requirements of the U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory authorities.

[0199] (v) Immune cell sample collection and cell enrichment. The types of immune cells are described above. This disclosure describes cells that have been genetically modified to express recombinant proteins such as bispecific antibodies. Cells genetically modified in accordance with the teachings of this disclosure may be patient-derived (autologous) or, where appropriate, allogeneic.

[0200] Methods for sample collection and concentration are known to those skilled in the art. In some embodiments, the cells are derived from cell lines. In some embodiments, the cells are obtained from heterologous sources, such as mice, rats, non-human primates, or pigs. In certain embodiments, the cells are derived from humans.

[0201] In some embodiments, T cells are derived from or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breasts, bones, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom. In certain embodiments, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In certain embodiments, the sample contains lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, HSCs, HPCs, HSPCs, erythrocytes and / or platelets, and in some embodiments, it contains cells other than erythrocytes and platelets and requires further processing.

[0202] In some embodiments, blood cells collected from the subject are washed, for example, to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing steps. In certain embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution is deficient in calcium and / or magnesium and / or many or all divalent cations. Washing may be achieved using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) may also be performed. In certain embodiments, after washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca++ / Mg++-free PBS.

[0203] Isolation may comprise one or more of various cell preparation and separation steps, including separation based on one or more properties such as size, density, sensitivity or tolerance to a particular reagent and / or affinity, e.g., immunoaffinity to an antibody or other binding partner. In certain embodiments, isolation is performed sequentially and / or simultaneously in a single process stream using the same apparatus or equipment. In certain embodiments, isolation, culture, and / or operations of different populations are performed from the same starting composition or material, such as from the same sample.

[0204] In certain embodiments, a sample may be enriched for T cells by using density-based cell separation methods and related techniques. For example, leukocytes may be separated from other cell types in peripheral blood by lysing erythrocytes and centrifuging the sample using a Percoll or Ficoll gradient.

[0205] In certain embodiments, a bulk T cell population that is not enriched for a particular T cell type may be used. In certain embodiments, the selected T cell type may be enriched and / or isolated based on positive and / or negative selection based on a cell marker. In positive selection, cells bound to the cell marker are retained for further use. In negative selection, cells not bound to the cell marker by a capture agent such as an antibody are retained for further use. In some examples, both fractions may be retained for further use.

[0206] Isolation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0207] In some cases, multiple rounds of isolation processes are performed, and fractions selected as positive or negative in one process are subjected to other isolation processes, such as subsequent positive or negative selection.

[0208] In some embodiments, antibodies or binding domains for cell markers bind to a solid support or matrix, such as magnetic or paramagnetic beads, to enable the separation of cells for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (as outlined in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17–25, edited by SABrooks and U.Schumacher (copyright) Humana Press Inc., Totowa, NJ). See also US4,452,773, US4,795,698, US5,200,084 and EP452342.

[0209] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The MACS system enables high-purity selection of cells to which magnetized particles are attached. In certain embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells attached to magnetized particles are retained in place, while non-attached species are eluted. Next, after this initial elution step is complete, species that were trapped by the magnetic field and whose elution was prevented are released in some way so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from heterogeneous cell populations.

[0210] In some embodiments, the cell populations described herein are collected and concentrated (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid flow. In some embodiments, the cell populations described herein are collected and concentrated (or depleted) by preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and concentrated (or depleted) by the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, e.g., WO2010 / 033140, Cho et al. (2010) Lab Chip 10, pp. 1567-1573; and Godin et al. (2008) J Biophoton. 1(5): pp. 355-376). In either case, cells may be labeled with multiple markers, enabling highly purified isolation of clearly defined cell subsets.

[0211] Cell markers for different T cell subpopulations are described above. In certain embodiments, cells expressing a specific subpopulation of T cells, e.g., positive or high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4 and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0212] CD3+, CD28+ T cells can be positively selected and expanded using anti-CD3 / anti-CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0213] In certain embodiments, a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD8+ and CD4+ populations may be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more naive, memory, and / or effector T cell subpopulations.

[0214] In some embodiments, enrichment is performed for central memory T (TCM) cells. In certain embodiments, memory T cells are present in both CD8+ and CD62L subsets of peripheral blood lymphocytes. PBMCs may be enriched or depleted for the CD62L, CD8, and / or CD62L+CD8+ fractions, for example, by using anti-CD8 and anti-CD62L antibodies.

[0215] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127; in some embodiments, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, isolation of the enriched CD8+ population of TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CCR7, CD45RO, and / or CD62L. In one embodiment, enrichment of central memory T (TCM) cells is performed starting with a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some embodiments and sequentially in any order in others. In some embodiments, the same CD4 expression-based selection step used in preparing a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, thereby selectively retaining both positive and negative fractions from CD4-based isolation after one or more further positive or negative selection steps.

[0216] In certain cases, PBMC samples or other leukocyte samples are subjected to CD4+ cell selection, retaining both negative and positive fractions. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or RORl, and positive selection based on markers characteristic of central memory T cells such as CCR7, CD45RO and / or CD62L, with positive and negative selection performed in either order.

[0217] In certain embodiments, cell enrichment results in a bulk CD8+FACs-sorted cell population.

[0218] Other cell types can be enriched based on known marker profiles and techniques. For example, CD34+HSCs, HSPs, and HSPCs can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles connected to a magnetic cell separation device, such as the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0219] (vi) Genetically modifying a cell population to express a recombinant protein. Desired genes encoding recombinant proteins disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate-mediated transfection, infection with a viral or bacteriophage vector containing a gene sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217, pp. 599-618; Cohen et al., 1993, Meth. Enzymol. 217: pp. 618-644; Cline, 1985, Pharmac. Ther. 29: pp. 69-92), and can be used as long as the necessary development and physiological functions of the recipient cells are not unnecessarily disrupted. This technology provides a stable introduction of genes into cells so that the genes are expressible by the cells, preferably heritable in certain cases, and expressible by the offspring of these cells.

[0220] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotides or nucleotide sequences) that codes for a recombinant protein disclosed herein. This definition includes polymorphisms, mutations, and / or sequence variants of various sequences, such modifications which do not substantially affect the function of the coded CAR. The term “gene” may include regulatory regions such as promoters, enhancers, and termination regions, as well as coding sequences. The term may further include all introns and other DNA sequences spliced ​​from mRNA transcripts, along with variants arising from alternative splice sites. Gene sequences that code for a molecule may be DNA or RNA that directs the expression of a chimeric molecule. These nucleic acid sequences may be DNA strand sequences transcribed to RNA or RNA sequences translated to proteins. Nucleic acid sequences include both full-length nucleic acid sequences and incomplete-length sequences derived from full-length proteins. Sequences may also include degenerate codons of native sequences(s) that may be introduced to provide codon selection in a particular cell type. As will be understood by those skilled in the art, portions of complete gene sequences are referenced throughout this disclosure.

[0221] Gene sequences encoding recombinant proteins are provided herein and can be readily prepared by synthesis or recombination methods from related amino acid sequences and other descriptions provided herein. In embodiments, gene sequences encoding any of these sequences may also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence to provide easy excision and replacement of the sequence-encoding gene sequence with another gene sequence encoding a different sequence. In embodiments, the sequence-encoding gene sequences may be codon-optimized for expression in mammalian cells.

[0222] "Code" refers to the characteristic of a particular sequence of nucleotides in a gene, such as cDNA or mRNA, that serves as a template for the synthesis of other macromolecules, such as defined sequences of amino acids. Therefore, a gene codes for a protein if the transcription and translation of mRNA equivalent to a gene produces a protein in a cell or other biological system. A "protein-coding gene sequence" includes all nucleotide sequences that code for the same amino acid sequence(s) of substantially similar form and function, in degenerate form of each other.

[0223] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, cosmids, viruses, or phages. An "expression vector" is a vector that, when present in the appropriate environment, can direct the expression of proteins encoded by one or more genes carried by the vector.

[0224] "Lentivirus" refers to a genus of retroviruses that can infect both dividing and non-dividing cells. Some examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0225] A "retrovirus" is a virus that has an RNA genome. "Gamma retrovirus" refers to a genus of the family Retroviridae. Examples of gamma retroviruses include mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and reticuloendotheliosis virus.

[0226] Retroviral vectors (see Miller et al., 1993, Meth. Enzymol. 217: pp. 581-599) may be used. In such embodiments, the gene to be expressed is cloned into a retroviral vector for delivery to cells. In certain embodiments, the retroviral vector includes all the cis-acting sequences necessary for packaging and incorporating the viral genome, namely (a) long terminal repeats (LTRs) or parts thereof at each end of the vector, (b) primer binding sites for the synthesis of minus and plus strand DNA, and (c) packaging signals necessary for the uptake of genomic RNA into virions. Further details on retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6: pp. 291-302; Clowes et al., 1994, J. Clin. Invest. 93: pp. 644-651; Kiem et al., 1994, Blood 83: pp. 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: pp. 129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: pp. 110-114. Adenoviruses, adeno-associated viruses (AAVs), and alphaviruses may also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: pp. 499-503; Rosenfeld et al., 1991, Science 252: pp. 431-434; Rosenfeld et al., 1992, Cell 68: pp. 143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91: pp. 225-234; Walsh et al., 1993, Proc. Soc. Exp. Bioi. Med. 204: pp. 289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: pp. 673-686.Other methods of gene delivery include the use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8: pp. 351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63: pp. 607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6: pp. 78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75: pp. 267-282).

[0227] Numerous suitable viral vectors are available within the scope of this disclosure, including those identified for application in human gene therapy (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum. Genet. 2: 177). Methods for packaging cells to transduce viral particles containing transgenes into mammalian host cells using retroviral and lentiviral vectors are described, for example, US8, 119, 772; Walchli et al., 2011, PLoS One 6: 327930; Zhao et al., 2005, J. Immunol. 174: 4415; Engels et al., 2003, Hum. Gene Ther. 14: 1155; Frecha et al., 2010, Mol. Ther. 18: 1748; and Verhoeyen et al., 2009, Methods Mol. Biol. 506: 97. Retrovirus and lentivirus vector constructs and expression systems are also commercially available.

[0228] Targeted gene manipulation approaches may also be utilized. The CRISPR (clustered, regularly arranged short palindromic sequence repeats) / Cas (CRISPR-related protein) nuclease system is an engineered nuclease system used for bacterial-based gene manipulation. Information on the CRISPR-Cas system and its components can be found, for example, in US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641, as well as to these and related applications; as well as WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO 2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354 This is described in WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and related applications.

[0229] Certain embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind to and cleave DNA at specific locations. ZFNs are used to introduce double-strand breaks (DSBs) at specific sites within a DNA sequence, which allows ZFNs to target unique sequences within the genome in various different cells. For further information regarding ZFNs and useful ZFNs within the scope of the teachings of this disclosure, see, for example, US6,534,261;US6,607,882;US6,746,838;US6,794,136;US6,824,978;6,866,997;US6,933,113;6,979,539;US7,013,219;US7,030,215;US7,220,719;US7,241,573;US7,241,574;US7,585,849;US7,595,376;US6,903,185;US6,479,626;US2003 / 0232410 and US2009 / 0203140 and Gaj et al., Nat Methods, 2012, 9(8):805-807; Ramirez et al., Nucle Acids Res, 2012, 40(12):5560-558; Kim et al., Genome Res, 2012, 22(7):1327-133; Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Miller et al., Nature biotechnology 25, 778-785 (2007); Bibikova et al., Science 300, 764 (2003); Bibikova et al., Genetics 161, 1169-1175 (2002); Wolfe et al., Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim et al., Proceedings of the National Academy of Sciences of See The United States of America 93, pp. 1156–1160 (1996); and Miller et al., The EMBO journal 4, pp. 1609–1614 (1985).

[0230] In certain embodiments, activator-like effector nucleases (TALENs) may be used as gene editing agents. A TALEN refers to a fusion protein comprising an activator-like effector (TALE) DNA-binding protein and a DNA-cleavage domain. TALENs are used to edit genes and genomes by inducing two double-strand breaks (DSBs) within the DNA, which induce intracellular repair mechanisms. Generally, the two TALENs must bind to each side of the target DNA site, with the DNA-cleavage domains flanking each other, so that they dimerize and induce the DSBs. For further information on TALEN, see US8,440,431;US8,440,432;US8,450,471;US8,586,363; and US8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4:1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-807; Miller et al., Nature biotechnology 29, 143-148 (2011); Christian et al., Genetics 186, 757-761 (2010); Boch et al., Science See also Moscou and Bogdanove, Science 326, p. 1509–1512 (2009); and Science 326, p. 1501 (2009).

[0231] In certain embodiments, MegaTAL can be used as a gene editing agent. MegaTAL has an sc-rare cleavage nuclease structure in which TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that possess both DNA recognition and nuclease functions within the same domain. In contrast to TALENs, megaTAL requires only the delivery of a single peptide chain for functional activity.

[0232] Nanoparticles that result in selective in vivo gene modification of targeted cell types are described and may be used within the scope of the teachings of this disclosure. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110 and WO201822672.

[0233] (vii) Cell activation culture conditions. Cell populations may be incubated in a culture initiation composition to expand the genetically modified cell population. Incubation may be performed in a culture vessel such as a bag, cell culture plate, flask, chamber, chromatography column, crosslinked gel, crosslinked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial, or other container for culture or cell culture.

[0234] Culture conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, drugs, such as nutrients, amino acids, antibiotics, ions and / or stimulants such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other drugs designed to activate cells.

[0235] In some embodiments, incubation is carried out in accordance with techniques such as those described in US6,040,177, Klebanoff et al. (2012) J Immunother.35(9):651-660; Terakura et al. (2012) Blood.1:72-82 and / or Wang et al. (2012) J Immunother.35(9):689-701.

[0236] Exemplary culture media for culturing T cells include: (i) RPMI supplemented with non-essential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) RPMI with HEPES, 5-15% human serum, 1-3% L-glutamine, 0.5-1.5% penicillin / streptomycin, and 0.25 × 10⁻⁴ to 0.75 × 10⁻⁴ M β-mercaptoethanol; and (iii) 10% fetal bovine serum. (iv) RPMI-1640 supplemented with FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin and 100 mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin and 100 mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human type AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax (Gibco), and 2% N-acetylcysteine ​​(Sigma-Aldrich, St. Louis, MO). T cell culture media are also commercially available from Hyclone (Logan, UT). Further T cell activating components that can be added to such culture media are described in more detail below.

[0237] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to a culture initiation composition (for example, so that the resulting cell population contains at least 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population being expanded), and incubating the culture (for example, for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of 3000-3600 rad to inhibit cell division. In some embodiments, the feeder cells are added to the culture medium before the T cell population is added.

[0238] Optionally, incubation may further include the addition of EBV-transformed non-dividing lymphoblastoid cells (LCLs) as feeder cells. The LCLs may be irradiated with gamma rays in the range of 6,000 to 10,000 rads. In some embodiments, the LCL feeder cells are provided in any suitable amount, such as LCL feeder cells versus early T lymphocytes in a ratio of at least 10:1.

[0239] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, for example, at least 25°C, at least 30°C, or 37°C.

[0240] In certain embodiments, the conditions for T cell activation culture may include T cell stimulating epitopes. T cell stimulating epitopes include CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1BB (CD137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1R, LAT, LFA-1, LIGHT, MHCI, MHCII, NKG2D, OX40, ROR2, and RTK.

[0241] CD3 is a major signaling element of T cell receptors. As previously shown, CD3 is expressed on all mature T cells. In certain embodiments, the CD3-stimulating molecule (i.e., the CD3-binding domain) may be derived from OKT3 antibodies (US5,929,212; US4,361,549; ATCC® CRL-8001®; and see Arakawa et al., J. Biochem. 120, pp. 657-662 (1996)), 20G6-F3 antibodies, 4B4-D7 antibodies, 4E7-C9 antibodies, or 18F5-H10 antibodies.

[0242] In certain embodiments, the CD3-stimulating molecule may be included in the culture medium at a concentration of at least 0.25 or 0.5 ng / ml or 2.5 to 10 μg / ml. In certain embodiments, the CD3-stimulating molecule (e.g., OKT3) is utilized at a concentration of 5 μg / ml.

[0243] In certain embodiments, activating molecules associated with the avi tag may be biotinylated and bound to streptavidin beads. This approach could be used, for example, to create a removable T cell epitope-stimulated activation system.

[0244] Exemplary binding domains for CD28 may include or be derived from the TGN1412, CD80, CD86, or 9D7 antibodies. Further antibodies that bind to CD28 include 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, and CD28.3 (deposited as a synthetic single-stranded Fv construct under GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, July 15, 2003, Vol. 102, No. 2, pp. 564-570). Furthermore, 1YJD provides a crystalline structure of human CD28 complexed with the Fab fragment of a mitotic-promoting antibody (5.11A1). In certain embodiments, an antibody that does not compete with 9D7 is selected.

[0245] The 4-1BB binding domain may be derived from LOB12, IgG2a, LOB12.3, or IgG1, as described in Taraban et al., Eur J Immunol. December 2002;32(12):3617-27. In certain embodiments, the 4-1BB binding domain is derived from the monoclonal antibody described in US9,382,328. Further 4-1BB binding domains are described in US6,569,997, US6,303,121, and Mittler et al., Immunol Res. 2004;29(1-3):197-208.

[0246] OX40 (CD134) and / or ICOS activation may also be used. The OX40 binding domain is described in US20100196359, US20150307617, WO2015 / 153513, WO2013 / 038191 and Melero et al., Clin Cancer Res. March 1, 2013;19(5):1044-53. Exemplary binding domains that can bind to and activate ICOS are described, for example, in US20080279851 and Deng et al., Hybrid Hybridomics. June 2004;23(3):176-82.

[0247] In its soluble form, the T-cell activator can be coupled with another molecule, such as polyethylene glycol (PEG) molecules. Any suitable PEG molecule can be used. Typically, PEG molecules with molecular weights up to 1000 Da are soluble in water or culture medium. In some cases, such PEG-based reagents can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives available from NOF North America Corporation, Irvine, Calif., USA, or activated PEG derivatives available from Creative PEGWorks, Chapel Hills, NC, USA).

[0248] In certain embodiments, the cell stimulant is immobilized on a solid phase within the culture medium. In certain embodiments, the solid phase is the surface of a culture vessel (e.g., a bag, cell culture plate, chamber, chromatography column, crosslinked gel, crosslinked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial, other structure or container for culture or cell culture).

[0249] In certain embodiments, a solid phase may be added to the culture medium. Such solid phases may include, for example, beads, hollow fibers, resins, membranes, and polymers.

[0250] Exemplary beads include magnetic beads, polymer beads, and resin beads (e.g., Strep-Tactin® Sepharose, Strep-Tactin® Superflow, and Strep-Tactin® MacroPrep IBA GmbH, Gottingen). Anti-CD3 / anti-CD28 beads are commercially available reagents for T cell expansion (Invitrogen). These beads are uniform, 4.5 μm superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of affinity-purified monoclonal antibodies against CD3 and CD28 cell surface molecules on human T cells. Hollow fibers are available from TerumoBCT Inc. (Lakewood, Colo, USA). Resins include metal affinity chromatography (IMAC) resins (e.g., TALON® resin (Westburg, Leusden)). The membrane includes paper and chromatography matrix membrane substrates (e.g., nitrocellulose membrane or polyvinylidene fluoride (PVDF) membrane).

[0251] Exemplary polymers include polysaccharides such as polysaccharide matrices. Such matrices include agarose gels (e.g., Superflow® Agarose or Sepharose® materials, e.g., Superflow® Sepharose®, commercially available in different bead sizes and pore sizes) or cross-linked dextran gels. A further exemplary example is a particulate cross-linked agarose matrix in which dextran is covalently bonded, commercially available as Sephadex® or Superdex® (in various bead sizes and pore sizes), both of which are available from GE Healthcare.

[0252] Possible synthetic polymers include copolymers of polyacrylamide, polymethacrylate, polysaccharides, and agarose (e.g., polyacrylamide / agarose composites) or polysaccharides and N,N'-methylenebisacrylamide. Examples of copolymers of dextran and N,N'-methylenebisacrylamide are the Sephacryl® (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) series materials.

[0253] Certain embodiments may utilize silica particles coupled to synthetic or natural polymers, such as polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethyl aspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

[0254] Cell activators can be immobilized on a solid phase via covalent bonds or reversibly immobilized via non-covalent bonds.

[0255] In certain embodiments, the T cell activation culture medium consists of HEPES, 5-15% human serum, 1-3% L-glutamine, 0.5-1.5% Pen / strep, and 0.25 × 10⁻¹⁴ cells. -4 ~0.75 × 10 -4The culture is performed on flat-bottom well plates plated with 0.1–0.5 × 10 e6 cells / well. The cells are transferred to TC-treated plates on day 3 after activation.

[0256] In certain embodiments, the T cell activation culture medium consists of HEPES, 10% human serum, 2% L-glutamine, 1% Pen / strep, and 0.5 × 10⁶ cells. -4 The cultures consist of individually cultured FACS-selected CD8+ T populations containing 5–15 (e.g., 10) ng / μl of IL-7, IL-15, and IL-21 in RPMI containing M β-mercaptoethanol. The cultures are performed on flat-bottom non-tissue culture (TC) treated 96 / 48 well plates plated with 0.1–0.5 × 10 e6 cells / well. Three days post-activation, the cells are transferred to TC-treated plates.

[0257] Culture conditions for HSC / HSP may include growth factors present under culture conditions such as those described below, including those expanded with Notch agonists (see, for example, US7,399,633; US5,780,300; US5,648,464; US5,849,869; and US5,856,441): 25-300 ng / ml of SCF, 25-300 ng / ml of Flt-3L, 25-100 ng / ml of TPO, 25-100 ng / ml of IL-6, and 10 ng / ml of IL-3. In more specific embodiments, 50, 100, or 200 ng / ml of SCF; 50, 100, or 200 ng / ml of Flt-3L; 50, or 100 ng / ml of TPO; 50, or 100 ng / ml of IL-6; and 10 ng / ml of IL-3.

[0258] (viii) Cell formulations manufactured ex vivo. In certain embodiments, genetically modified cells may be harvested from culture medium, washed, and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0259] In certain embodiments, the carrier may be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In certain embodiments, the carrier for infusion may contain buffered saline with 5% HAS or dextrose. Further isotonic agents may include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols, including glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0260] The carrier may include buffers such as citrate buffer, succinate buffer, tartarate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer, and / or trimethylamine salts.

[0261] Stabilizers refer to a broad range of excipients, ranging in function from bulking agents to additives that help prevent cells from adhering to container walls. Typical stabilizers include polyhydric sugar alcohols; amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, and cyclitol, e.g., inositol; PEG; amino acid polymers; sulfur-containing reducing agents, e.g., urea, glutathione. These may include: onions, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., less than 10 residues); proteins, e.g., HSA, bovine serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; monosaccharides, e.g., xylose, mannose, fructose, and glucose; disaccharides, e.g., lactose, maltose, and sucrose; trisaccharides, e.g., raffinose; and polysaccharides, e.g., dextran.

[0262] Where necessary or beneficial, the composition or formulation may contain a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0263] Exemplary preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0264] The therapeutically effective amount of cells in the composition or preparation is 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than one cell or 10 11 It can exceed the individual.

[0265] In the compositions and formulations disclosed herein, the cells are generally in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the density of administered cells is typically 10 4 cells / ml, 10 7 cells / ml or 10 8 More than individual cells / ml.

[0266] As shown, the composition comprises at least one genetically modified cell type (e.g., modified T cells, NK cells, or stem cells). The formulation may comprise different types of genetically modified cells (e.g., T cells, NK cells, and / or combined stem cells).

[0267] Different types of genetically modified cells or cell subsets (e.g., modified T cells, NK cells, and / or stem cells) can be provided in different ratios, such as 1:1:1, 2:1:1, 1:2:1, 1:1:2, 5:1:1, 1:5:1, 1:1:5, 10:1:1, 1:10:1, 1:1:10, 2:2:1, 1:2:2, 2:1:2, 5:5:1, 1:5:5, 5:1:5, 10:10:1, 1:10:10, 10:1:10, etc. These ratios can also be applied to the number of cells expressing the same or different recombinant proteins. If only two of the cell types are combined or only two combinations of recombinant proteins are included in the formulation, the ratio may include any two combinations of numbers that can be made from the three combinations of numbers provided above. In embodiments, the combined cell population is tested in vitro, in vivo and / or ex vivo for efficacy and / or cell proliferation to select a cell ratio that results in cell efficacy and / or proliferation. A particular embodiment includes CD4 T cells and CD8 T cells in a 1:1 ratio.

[0268] The cell-based compositions disclosed herein may be prepared, for example, for administration by injection, infusion, perfusion, or lavage. The compositions and formulations may be further formulated for intramedullary, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intrafocal, intratumoral, intrabladder, and / or subcutaneous injection.

[0269] (ix) Method of use. The methods disclosed herein include treating subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle, goats, pigs, sheep, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.) with the compositions disclosed herein. Treating a subject includes delivering a therapeutically effective dose. A therapeutically effective dose includes an effective dose, a prophylactic dose and / or a therapeutic dose.

[0270] An "effective dose" is the amount of a composition required to produce a desired physiological change in a subject. Effective doses are often administered for research purposes. The effective doses disclosed herein may produce statistically significant effects in animal models or in vitro assays related to the assessment of the onset or progression of CD33-related disorders.

[0271] "Prophylactic measures" include measures administered to subjects who do not show signs or symptoms of CD33-related (e.g., CD33 expression) disorder, or who show only early signs or symptoms of CD33-related disorder, thereby reducing or decreasing the risk of further progression of the CD33-related disorder. Therefore, prophylactic measures function as measures to prevent CD33-related disorder.

[0272] "Therapeutic treatment" may include treatment administered to subjects exhibiting signs or symptoms of CD33-related disorder, with the aim of reducing or eliminating those signs or symptoms of CD33-related disorder. Therapeutic treatment may reduce, control or eliminate the presence or activity of CD33-related disorder, and / or reduce, control or eliminate the side effects of CD33-related disorder.

[0273] "Therapeutic procedures" may also include procedures administered to subjects requiring imaging. Subjects may require imaging to aid in diagnosis, to locate therapeutic interventions, to assess the function of body parts, and / or to assess the presence or absence of a condition. The effectiveness of therapeutic imaging procedures may be confirmed based on sufficient image acquisition for their intended purpose. Exemplary types of imaging include positron emission tomography (PET), single-photon emission computed tomography, radioisotope lenography, and scintigraphy.

[0274] The functions of an effective dose, prophylactic treatment, or therapeutic treatment are not mutually exclusive, and in certain embodiments, the administered dose may achieve two or more types of treatment.

[0275] In certain embodiments, a therapeutically effective dose produces an anticancer effect. The anticancer effect includes a reduction in the number of cancer cells, a reduction in the number of metastases, prevention or reduction of metastasis, a reduction in tumor volume, inhibition of tumor growth, extension of life expectancy, extension of the lifespan of the subject, induction of chemosensitivity or radiosensitivity in cancer cells, inhibition of cancer cell proliferation, reduction of cancer-related pain, and / or reduction of cancer recurrence or relapse after treatment.

[0276] A "tumor" is a swelling or lesion formed by the abnormal growth of cells (called neoplastic cells or tumor cells). "Tumor cells" are abnormal cells that grow through rapid, uncontrolled proliferation and continue to grow even after the initial stimulus that initiated their growth has ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue and typically form a distinct tissue mass that can be benign, premalignant, or malignant.

[0277] In certain embodiments, a therapeutically effective dose induces an immune response. This immune response may be against cancer cells.

[0278] Examples of CD33-related disorders include hematological cancers such as leukemia and lymphoma, and other myeloid or lymphoproliferative disorders.

[0279] Exemplary leukemias include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), mast cell leukemia, myelodysplastic syndrome (MDS), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and megakaryocytic leukemia.

[0280] Exemplary subtypes of AML include acute basophilic leukemia, acute erythroleukemia (AML-M6), acute megakaryoblastic leukemia (AML-M7), acute monoblastic leukemia (AML-M5a), acute monocytic leukemia (AML-M5b), acute myeloblastic leukemia with granulocyte maturation, acute myeloblastic leukemia without maturation, acute myelomonocytic leukemia (AML-M4), acute panmyelopathy with myelofibrosis, acute promyelocytic leukemia (APL), erythroleukemia (AML-M6a), minimally differentiated acute myeloblastic leukemia, myelomonocytic leukemia with myeloeosinophilia, and pure erythroleukemia (AML-M6b).

[0281] Exemplary lymphomas include multiple myeloma.

[0282] The compositions disclosed herein may also be used to treat complications or diseases associated with the lymphoproliferative disorders and hematological malignancies described above. For example, complications associated with AML may include preceding myelodysplastic syndromes (MDS, formerly known as “preleukemia”), secondary leukemia, particularly secondary AML, leukocytosis, and Auer's body deficiency. In particular, leukopenia and central nervous system (CNS) involvement, leukocytosis, and residual disease are also considered complications or diseases associated with AML.

[0283] The compositions disclosed herein may be used to target myeloid-derived suppressor cells (MDSCs). MDSCs are major players in the immunosuppressive tumor microenvironment and have been found to inhibit the antitumor responsiveness of T cells and NK cells. Certain MDSCs, including monocytic and immature MDSCs, have high CD33 expression and may be targeted for anti-CD33 treatment.

[0284] The compositions disclosed herein may also be found to be used in the treatment of other conditions or genetic syndromes associated with the risk of AML, such as Down syndrome, trisomy, Fanconi anemia, Bloom syndrome, ataxia telangiectasia, Diamond-Blackfan anemia, Schwachmann-Diamond syndrome, Li-Fraumeni syndrome, neurofibromatosis type 1, and severe congenital neutropenia (also known as Kostman syndrome).

[0285] The compositions disclosed herein may also be found to be used in the treatment of Alzheimer's disease.

[0286] With regard to administration, a therapeutically effective dose (also referred to herein as dose) may be initially estimated based on results from in vitro assays and / or animal model studies. Such information may be used to more accurately determine an effective dose in the subject of interest. The actual dose administered to a particular subject may be determined by a physician, veterinarian, or researcher, taking into account parameters such as physical and physiological factors, including the target, body weight, severity of the condition, type of CD33-related disorder, stage of the CD33-related disorder, past or concomitant therapeutic interventions, the subject's idiopathic disease, and route of administration.

[0287] Useful doses may range from 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In other examples, doses may include 1 μg / kg, 15 μg / kg, 30 μg / kg, 50 μg / kg, 55 μg / kg, 70 μg / kg, 90 μg / kg, 150 μg / kg, 350 μg / kg, 500 μg / kg, 750 μg / kg, 1000 μg / kg, 0.1 to 5 mg / kg, or 0.5 to 1 mg / kg. In other examples, doses may include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or higher.

[0288] A therapeutically effective amount of cell-based composition is 10 4 ~10 9 cells / kg body weight or 10 3 ~1011 May contain cells / kg body weight. The therapeutically effective dose to administer is 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than one cell or 10 11 It may include more than one individual.

[0289] The therapeutically effective dose may be achieved by administering one or more doses (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or annually) during the course of the treatment regimen. In certain embodiments, the treatment protocol may be directed by a clinical trial protocol or an FDA-approved treatment protocol.

[0290] The pharmaceutical compositions described herein may be administered by injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration may include intravenous, intradermal, intra-arterial, parenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intrafocal, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, oral, subcutaneous, and / or sublingual administration, and more specifically, intravenous, intradermal, intra-arterial, parenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intrafocal, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, oral, subcutaneous, and / or sublingual injection.

[0291] The methods of use also include, for example, the use of the antibodies described herein in image-based diagnostics when the antibodies are formulated as radioisotope conjugates.

[0292] (x) Reference levels derived from a control population. Values ​​obtained for parameters relating to the treatments described herein may be compared to reference levels derived from a control population, and this comparison may indicate whether the treatments described herein are effective for subjects requiring them. Reference levels may be obtained from one or more relevant datasets from a control population. As used herein, “dataset” is a set of numerical values ​​obtained from the evaluation of a sample (or a group of samples) under desired conditions. The values ​​of a dataset may be obtained, for example, by experimentally obtaining measurements from a sample and constructing a dataset from these measurements. As will be understood by those skilled in the art, reference levels may be based on any known mathematical or statistical formula, e.g., mean, median, median of means, etc., which is useful in the art to arrive at a meaningful collective reference level from a collection of individual data points. Alternatively, reference levels or datasets for creating reference levels may be obtained from a service provider such as a laboratory, or from a database or server on which datasets are stored.

[0293] The reference level from the dataset may be derived from previous measurements taken from a control population. “Control population” is any classification of subjects or samples with similar specific characteristics. Classification may follow, for example, clinical parameters, clinical assessments, treatment regimens, disease status, severity of condition, etc. In certain embodiments, classification is based on age range (e.g., 60–65 years) and non-immunocompromised status. In certain embodiments, the normal control population includes individuals who are age-matched with the test subjects and are not immunocompromised. In certain embodiments, age-matching may include, for example, 0–10 years, 30–40 years, 60–65 years, 70–85 years, etc., to be clinically relevant under the context. In certain embodiments, the control population may include a control population that has CD33-related impairment and has never been administered a therapeutically effective dose of the composition or formulation.

[0294] In certain embodiments, relevant reference levels for values ​​of specific parameters related to the treatments described herein are obtained based on values ​​of specific corresponding parameters related to the treatment in a control population to determine whether the treatment disclosed herein is therapeutically effective for subjects requiring it.

[0295] In certain embodiments, conclusions are drawn based on whether the sample value is statistically significantly different from or not statistically different from a reference level. A measurement is not statistically significant if the difference falls within the range of levels expected to occur solely by chance. In contrast, a statistically significant difference or increase is greater than what would be expected to occur solely by chance. Statistical significance or lack thereof can be determined by any of the various methods known in the art. An example of a commonly used measure of statistical significance is the p-value. The p-value represents the probability of obtaining a given result corresponding to a particular data point, where the data point is the result of random chance alone. Results are often considered significant (not random by chance) with a p-value of 0.05 or less. In certain embodiments, if the sample value and the reference level are not statistically significantly different, the sample value is "equivalent to" the reference level derived from a normal control population.

[0296] The following exemplary embodiments and examples are included to demonstrate certain non-limiting embodiments of the Disclosure. Those skilled in the art will recognize that, in light of the Disclosure, many modifications can be made to the specific embodiments disclosed herein without departing from the spirit and scope of the Disclosure, and similar or comparable results can still be obtained.

[0297] (xi) Exemplary embodiment.

[0298] 1. An antibody or antigen-binding fragment thereof containing a complementation-determining region (CDR) of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 according to North, IMGT, Kabat, Chothia, or Set5.

[0299] 2.1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 variable light chains and variable heavy chains, Variable light chains having at least 90% sequence identity with the variable light chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3, and variable heavy chains having at least 90% sequence identity with the corresponding variable heavy chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3. An antibody containing an antigen-binding fragment thereof.

[0300] 3. The antibody or antigen-binding fragment of Embodiment 1 or 2, wherein the antigen-binding fragment comprises VH-VL or VL-VH oriented Fv, Fab, Fab', F(ab')2, or single-stranded Fv fragment (scFv) (see, for example, SEQ ID NOs. 230-237 in Figure 14).

[0301] 4. An antibody or antigen-binding fragment of any of Embodiments 1 to 3, wherein the antibody or its antigen-binding fragment is PEGylated.

[0302] 5. An antibody according to any of Embodiments 1 to 4, or an antigen-binding fragment thereof, comprising an Fc modification.

[0303] 6. The antibody or antigen-binding fragment of Embodiment 5, wherein the Fc modification includes the M428L / N434S, G236A / S239D / A330L / I332E (GASDALIE), huIgG4 ProAlaAla, huIgG2m4, and / or huIgG2sigma mutations.

[0304] 7. An antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 6, which binds to the C2-set Ig-like domain of CD33 within 115 residues of the transmembrane region, regardless of the presence of the V-set domain, or which binds to the C2-set Ig-like domain only in the absence of the V-set domain.

[0305] 8. CD33 targeting agents comprising a binding domain containing a set of complementarity-determining regions (CDRs) of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 according to North, IMGT, Kabat, Chothia, or Set5, as part of an anti-CD33 immunotoxin, anti-CD33 antibody-drug conjugate, anti-CD33 antibody-fluorophore conjugate, anti-CD33 antibody-radioisotope conjugate, anti-CD33 bispecific antibody, anti-CD33 bispecific immune cell-inducing antibody, anti-CD33 tripspecific antibody, and / or anti-CD33 quadruspecific antibody.

[0306] 9. Anti-CD33 immunotoxins, anti-CD33 antibody-drug conjugates, anti-CD33 antibody-fluorophore conjugates, anti-CD33 antibody-radioisotope conjugates, anti-CD33 bispecific antibodies, anti-CD33 bispecific immune cell-inducing antibodies, anti-CD33 trispecific antibodies, and / or as part of anti-CD33 tetraspecific antibodies, variable weights and variable light chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3; or Variable weights and light chains having at least 90% sequence identity to the corresponding variable weights and light chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 as part of an anti-CD33 immunotoxin, anti-CD33 antibody-drug conjugate, anti-CD33 antibody-fluorophore conjugate, anti-CD33 antibody-radioisotope conjugate, anti-CD33 bispecific antibody, anti-CD33 bispecific immune cell inducing antibody, anti-CD33 tripspecific antibody, and / or as part of an anti-CD33 quadruspecific antibody. A CD33 targeting agent containing a binding domain.

[0307] 10. The CD33 targeting agent according to Embodiment 8 or 9, wherein the CD33 targeting agent comprises an anti-CD33 immunotoxin, and the toxin comprises a holotoxin or a hemitoxin.

[0308] 11. A CD33 targeting agent according to any one of Embodiments 8 to 10, wherein the CD33 targeting agent comprises an anti-CD33 immunotoxin, the toxin comprising abrin, bougain, bryodin 1, diphtheria toxin (DT), geronin, pygmyx lectin, modecin, pokeweed antiviral protein (PAP), pseudomonas exotoxin (PE), lysine and / or saporin.

[0309] 12. CD33 targeting agents include anti-CD33 antibody-drug conjugates, and the drugs include monomethyl auristatin E [MMAE], vedotin, dorastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin, PNU-159682, anthracycline, duocalmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, erinafide, taxol, cytochalasin B, gramicin A CD33 targeting agent according to any one of Embodiments 8 to 11, comprising din D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, and / or propranolol.

[0310] 13. A CD33 targeting agent according to any one of Embodiments 8 to 12, wherein the CD33 targeting agent comprises an anti-CD33 antibody-radioisotope conjugate, the radioisotope comprising arsenic-72, arsenic-74, iodine-131, indium-111, yttrium-90, lutetium-177, astatine-211, actinium-225, or bismuth-212 and / or bismuth-213.

[0311] 14. The CD33 targeting agent contains an anti-CD33 antibody-radioisotope conjugate, and the radioisotope is 225 Ac, 228 Ac, 111 Ag, 124 Am,74 As、 211 Available、 209 Available、 194 I、 128 They、 7 Be、 206 Wind、 245 Bk、 246 Bk、 76 Br、 11 C、 47 The、 254 Cf、 242 Cm、 51 Cr、 67 Cu、 153 Of, 157 Of, 159 Of, 165 Of, 166 Of, 171 On, 250 I、 254 I、 147 Eu、 157 Eu、 52 Faith、 59 Faith、 251 Fm、 252 Fm、 253 Fm、 66 Though, 72 Though, 146 Gd、 153 Gd、 68 Ge、 170 Hf、 171 Hf、 193 Hg、 193 mHg、 160 mHo、 130 I、 131 I、 135 I、 114 mIn、 185 Is、 42 K、 43 K、 76 Kr、 79 Kr、 81 mKr、 132 The、 262 Lr、 169 Lu、 174 mLu、 176 mLu、 257 Eg、 260 Eg、 28 Mg、 52 Mn、 90 For、 24 Than, 95Nb, 138 Nd, 57 Ni, 66 Ni, 234 Np, 15 O, 182 Os, 189 mOs, 191 Os, 32 P, 201 Pb, 101 Pd, 143 Pr, 191 Pt, 243 Pu, 225 Ra, 81 Rb, 188 Re, 105 Rh, 211 Rn, 103 Ru, 35 S, 44 Sc, 72 Se, 153 Sm, 125 Sn, 91 Sr, 173 Ta, 154 Tb, 127 Te, 234 Th, 45 Ti, 166 Tm, 230 U, 237 U, 240 U, 48 V, 178 W, 181 W, 188 W, 125 Xe, 127 Xe, 133 Xe, 133 mXe, 135 Xe, 85 mY, 86 Y, 90 Y, 93 Y, 169 Yb, 175 Yb, 65 Zn, 71 mZn, 86 Zr, 95 Zr and / or 97 A CD33 targeting agent according to any one of embodiments 8 to 13, comprising Zr.

[0312] 15. A CD33 targeting agent according to any one of embodiments 8 to 14, comprising a multispecific antibody.

[0313] 16. The CD33 targeting agent according to Embodiment 15, wherein the multispecific antibody comprises a bispecific antibody, a triplicate antibody, or a quadruplicate antibody.

[0314] 17. A CD33 targeting agent according to Embodiment 15 or 16, wherein the multispecific antibody includes a binding domain that activates immune cells (see, for example, SEQ ID NOs. 157 and 238-245 in Figure 14).

[0315] 18. The CD33 targeting agent according to Embodiment 17, wherein the immune cells are T cells, natural killer (NK) cells, NK-T cells, or macrophages.

[0316] 19. The CD33 targeting agent according to Embodiment 18, wherein the T cells are CD3 T cells, CD4 T cells, CD8 T cells, central memory T cells, effector memory T cells and / or naive T cells.

[0317] 20. A CD33 targeting agent according to any one of Embodiments 17 to 19, wherein the binding domain that activates immune cells binds to CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Rα, or MARCO.

[0318] 21. A CD33 targeting agent according to any of Embodiments 17 to 20, wherein the binding domain activates T cells and comprises a CDR of OKT3 antibody, 4B4-D7 antibody, 4E7-C9 antibody, 18F5-H10 antibody, or CD3 HcFv and CD3 LcFv as shown in SEQ ID NOs. 161 and 162.

[0319] 22. A CD33 targeting agent according to any of Embodiments 17 to 21, wherein the binding domain activates T cells and comprises a CDR of the TGN1412 antibody.

[0320] 23. A CD33 targeting agent according to any of Embodiments 17 to 22, wherein the binding domain activates T cells and contains a CDR of an OKT8 antibody.

[0321] 24. A CD33 targeting agent according to any of Embodiments 17 to 23, wherein the binding domain activates T cells and includes a TCR.

[0322] 25. CD33 targeting agent, A CDR set of 1H10, and CD3 HcFv having the sequence described in SEQ ID NO: 161 and CD3 LcFv having the sequence described in SEQ ID NO: 162; CD3 HcFv having the sequence described in 1A9 and CD3 LcFv having the sequence described in SEQ ID NO: 161; A 1E6 CDR set, and CD3 HcFv having the sequence described in SEQ ID NO: 161 and CD3 LcFv having the sequence described in SEQ ID NO: 162; A 1D2 CDR set, and CD3 HcFv having the sequence described in Sequence ID No. 161 and CD3 LcFv having the sequence described in Sequence ID No. 162; CD3 HcFv having the sequence described in 1B9 and sequence described in SEQ ID NO: 161 and CD3 LcFv having the sequence described in SEQ ID NO: 162; A 1H8 CDR set, and CD3 HcFv having the sequence described in SEQ ID NO: 161 and CD3 LcFv having the sequence described in SEQ ID NO: 162; A 2D3 CDR set and CD3 HcFv having the sequence described in Sequence ID No. 161 and CD3 LcFv having the sequence described in Sequence ID No. 162; or CD3 HcFv having the sequence described in Sequence ID No. 161 and CD3 LcFv having the sequence described in Sequence ID No. 162; A CD33 targeting agent having any of embodiments 17 to 24.

[0323] 26. A CD33 targeting agent according to any one of Embodiments 17 to 25, wherein the CD33 targeting agent has the sequence described in Sequence ID No. 234 or 235.

[0324] A CD33 targeting agent according to any of Embodiments 8 to 26, comprising 27.1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 Fv, Fab, Fab', F(ab')2, or a single-stranded Fv fragment (scFv), wherein the scFv may be VH-VL oriented or VL-VH oriented (see, for example, SEQ ID NOs. 230-237 in Figure 14).

[0325] 28. An antibody or antigen-binding fragment thereof according to any of Embodiments 1 to 7, or a CD33 targeting agent according to any of Embodiments 8 to 27, further comprising a linker.

[0326] 29. A CD33 targeting agent of Embodiment 28, wherein the linker is a Gly-Ser linker.

[0327] 30. Gly-Ser linker, (Gly x Ser y ) n A CD33 targeting agent according to Embodiment 29, comprising, where x and y are independently integers from 0 to 10, provided that both x and y are not 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0328] 31. A CD33 targeting agent according to Embodiment 30, wherein the Gly-Ser linker comprises (Gly4Ser)4 (SEQ ID NO: 125), (Gly4Ser)3 (SEQ ID NO: 126), (Gly4Ser)2 (SEQ ID NO: 127), (Gly4Ser)1 (SEQ ID NO: 128), (Gly3Ser)2 (SEQ ID NO: 129), (Gly3Ser)1 (SEQ ID NO: 130), (Gly2Ser)2 (SEQ ID NO: 131), (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 132), GGSGGGSGSG (SEQ ID NO: 133), or GGSGGGSG (SEQ ID NO: 134).

[0329] 32. A composition comprising an antibody or antigen-binding fragment thereof from any of Embodiments 1 to 7 or 28, and / or a CD33 targeting agent from any of Embodiments 8 to 31, formulated for administration to a target.

[0330] 33. Cells genetically modified to express an antibody or antigen-binding fragment thereof from any of Embodiments 1 to 7 and / or a CD33 targeting agent from any of Embodiments 8, 9, or 15 to 31, formulated for administration to a subject.

[0331] 34. The cells according to Embodiment 33, which are in vivo or ex vivo.

[0332] 35. The cell according to Embodiment 33 or 34, which is a T cell, B cell, natural killer (NK) cell, NK-T cell, monocyte / macrophage, hematopoietic stem cell (HSC), or hematopoietic progenitor cell (HPC).

[0333] 36. A cell according to any one of embodiments 33 to 35, which is a T cell selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells.

[0334] 37. A cell according to any of embodiments 33 to 36, wherein the cell is a CD8+ T cell.

[0335] 38. A formulation comprising a population of cells according to any of embodiments 33 to 37 and a pharmaceutically acceptable carrier.

[0336] 39. A method for treating a CD33-related disorder in a subject requiring the treatment thereof, comprising administering a therapeutically effective amount of the composition and / or the formulation described in Embodiment 32 and / or the formulation described in Embodiment 38 to the subject, thereby treating a CD33-related disorder in the subject requiring the treatment thereof.

[0337] 40. The method according to Embodiment 39, wherein CD33-related disorders include acute myeloid leukemia (AML).

[0338] 41. The method according to Embodiment 39, wherein the CD33-related disorder includes acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), mast cell leukemia, myelodysplastic syndrome (MDS), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or megakaryocytic leukemia.

[0339] 42. Any method according to Embodiments 39 to 41, wherein the population of cells in the formulation is autologous or homogeneous to the subject.

[0340] 43. To determine whether the subject expresses or lacks the V set domain of CD33, and If the target organism expresses the V set domain of CD33, One or more binding domains among 1H10, 1A9, 1E6, 1D2, and 1B9, and One or more binding domains from 1H8, 2D3, and 2E3 The method according to any one of embodiments 39 to 42, further comprising selecting a combination therapy comprising a composition comprising the above.

[0341] 44. To determine whether the subject expresses or lacks the V set domain of CD33, and If the subject does not express the V set domain of CD33, The method according to any one of embodiments 39 to 42, further comprising selecting a therapy comprising a composition containing one or more binding domains from among 6H9, 9G2, 3A5, 7D5, 1H7, and 2D5.

[0342] 45. A method for activating an immune response to CD33-expressing cells in a subject requiring such activation, comprising administering a therapeutically effective amount of the composition and / or the formulation described in Embodiment 38 to the subject.

[0343] 46. ​​Embodiment 45, in which CD33-expressing cells include acute myeloid leukemia (AML) cells. Methods used.

[0344] 47. The method according to Embodiment 45, wherein the CD33-expressing cells include cells of acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), mast cell leukemia, myelodysplastic syndrome (MDS), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or megakaryocytic leukemia.

[0345] 48. A method according to any of Embodiments 45 to 47, wherein the population of cells in the formulation is autologous or homogeneous to the subject.

[0346] 49. To determine whether the subject expresses or lacks the V set domain of CD33, and If the target organism expresses the V set domain of CD33, One or more binding domains among 1H10, 1A9, 1E6, 1D2, and 1B9, and Composition comprising one or more binding domains from among 1H8, 2D3, and 2E3 The method according to any one of embodiments 45 to 48, further comprising selecting a combination therapy including the following:

[0347] 50. To determine whether the subject expresses or lacks the V set domain of CD33, and If the subject does not express the V set domain of CD33, Composition comprising one or more binding domains from among 6H9, 9G2, 3A5, 7D5, 1H7, and 2D5 The method according to any one of embodiments 45 to 48, further comprising selecting a therapy that includes the following.

[0348] A kit comprising a composition containing one or more binding domains from among 51.1H10, 1A9, 1E6, 1D2, and 1B9, and one or more binding domains from among 1H8, 2D3, and 2E3.

[0349] A kit comprising a composition containing one or more binding domains from among 52.1H10, 1A9, 1E6, 1D2, and 1B9, and a composition containing one or more binding domains from among 1H8, 2D3, and 2E3.

[0350] 53. Use of an antibody or antigen-binding fragment thereof described in any of the prior embodiments, or a CD33 targeting agent described in any of the prior embodiments, for in vivo imaging and / or for enriching, isolating and / or detecting CD33-expressing cells in vitro or in vivo.

[0351] (xii) Experimental example. Summary. There is growing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, this strategy has been confirmed by the extension of survival with gemtuzumab ozogamicin (GO), a CD33 antibody-drug conjugate. Nevertheless, GO is beneficial only to some patients, prompting attempts to develop more potent CD33-directed therapies. One limitation is that CD33 antibodies typically recognize the distal V-set domain of the membrane. We investigated whether targeting a membrane-proximal targeting epitope using various synthetic CD33 proteins, in which this domain is located differently within the extracellular portion of the molecule, would enhance the effector function of CD33 antibody-based therapies. Consistent with this idea, CD33 Vセット The CD3 bispecific antibody (BsAb) induced substantially greater cytotoxicity in cells expressing a CD33 variant lacking the entire C2 set domain than in cells expressing full-length CD33, but the cytotoxic effect induced by GO was independent of the position of the V set domain. Therefore, mouse and human antibodies were produced against the C2 set domain of human CD33, and the antibodies bound to CD33 ("CD33") were independent of the presence or absence of the C2 set domain. PAN Antibodies were identified. These antibodies, upon binding to CD33, are internalized and then... PAN / CD3 BsAb, CD33 +This resulted in a strong cell-lysing effect on the cells. This data suggests that CD33 PAN This provides a theoretical basis for further developing antibody-based therapies.

[0352] Introduction. CD33 (Siglec-3) is a differentiation antigen primarily presented on mature and mature myeloid cells and their neonatal counterparts (Walter et al., Blood. 119(26): pp. 6198-6208, 2012; and Duan et al., Annu Rev Immunol. 38: pp. 365-395, 2020). This expression pattern has led to long-term attempts to therapeutically target CD33+ cells, primarily in acute myeloid leukemia (AML) (Walter et al., Blood. 119(26): pp. 6198-6208, 2012; Grossbard et al., Blood. 80(4): pp. 863-878, 1992; and Laszlo et al., Blood Rev. 28(4): pp. 143-153, 2014). Similarly, this has also been done in CD33+ tumor cells, CD33+ bone marrow-derived suppressor cells, and normal CD33+ microglia cells in other malignancies (Walter, Expert Opin Biol Ther. 20(9): pp. 955-958, 2020). In AML, the effectiveness of CD33 as a drug target has been confirmed by the fact that the survival time of some patients treated with the antibody-drug conjugate GO was extended (Godwin et al., Leukemia. 31(9): pp. 1855-1868, 2017).

[0353] The successes and limitations of GO have spurred ongoing research to develop more effective CD33-targeted therapies. However, targeting CD33 has proven challenging, and several drugs have failed clinically due to a lack of efficacy. Therefore, efforts have focused on developing more potent anti-CD33 therapies, including T cell-inducing bispecific antibodies (BsAbs). One significant limitation of these attempts is that existing and investigational therapies, including GO, almost exclusively recognize the immunodominant epitope within the membrane distal V-set domain encoded in exon 2 of CD33 (Figure 1) (Walter, Expert Opin Investig Drugs. 27(4):339-348, 2018). Since antibody membrane-proximal binding can enhance their effector function (Bluemel et al., Cancer Immunol Immunother. 59(8): pp. 1197-1209, 2010; Lin, Pharmgenomics Pers Med. 3: pp. 51-59, 2010; Haso et al., Blood. 121(7): pp. 1165-1174, 2013; Cleary et al., J Immunol. 198(10): pp. 3999-4011, 2017), targeting CD33 with antibodies against membrane-proximal C2 set domains should optimize CD33-directed therapies that induce immune effector cells. Here, this concept has been experimentally tested, and the creation of a series of C2 set domain-directed CD33 antibodies and therapies derived therefrom is described.

[0354] Results: The binding distance from the cell membrane correlates with the immunoeffector function of the CD33 antibody. To investigate whether the distance between the target epitope and the cell membrane affects the efficacy of T cell-inducing immunotherapy, a series of artificial proteins were generated in which the V-set domain of human CD33 was maintained at different distances from the cell membrane. VセットThis enabled targeting with V-set domain-oriented CD33 antibody-based therapies such as CD33 BsAb (Figure 2). Specifically, to bring the CD33 target epitope closer to the cell membrane, an artificial CD33 protein lacking the entire C2 set domain was created by removing exons 3 and 4 (CD33 ΔE3-4 This generated manipulated human CD33 cells in which endogenous CD33 was deleted by CRISPR / Cas9 (Humbert et al., Leukemia. 33(3):762-808, 2019). + Using AML cell lines, CD33 FL or CD33 ΔE3-4 In the initial series of experiments, sublines expressing relatively similar levels of the target molecule were subjected to various doses of CD33. Vセット CD33 BsAb and healthy donor T cells as immunoeffector cells were subjected to short-term in vitro cytotoxic assays. GO was used as a comparator, which relies entirely on the toxic effect induced by the calicheamicin-γ1 payload for its antitumor effect (Walter et al., Blood. 119(26): pp. 6198-6208, 2012; Laszlo et al., Blood Rev. 28(4): pp. 143-153, 2014; and Godwin et al., Leukemia. 31(9): pp. 1855-1868, 2017). As shown in Figures 3A-3C, CD33 Vセット / CD3 BsAb is CD33 FL Cells that express CD33 ΔE3-4 It exhibited significant cytotoxicity against AML and ALL cells expressing CD33, but the cytotoxic effect induced by GO was similar. Vセット When treated with CD3 BsAb, REH and RS4;11 cells expressing these same CD33 constructs (human CD33 -Similar effects were observed in the B acute lymphoblastic leukemia [B-ALL] cell line (data for RS4;11 are shown in Figure 4). To further demonstrate the importance of target epitope membrane distance for the efficacy of CD33-targeted therapy that induces T cells, chimeric proteins were generated using various parts of human CD22 to extend the distance between the CD33 target epitope and the cell membrane (Figure 2). As summarized in Figure 5, CD33 Vセット The cytotoxic effect of CD3 BsAb is due to CD33 FL The paired cells that express CD22 / CD33 FL The effect was lower in AML cells expressing the chimeric protein. In summary, these data demonstrate that altering the location of the CD33 antibody-binding epitope alters the effector function of CD33 antibody-derived therapies, suggesting that membrane-proximal targeting of CD33 via C2 set domain-specific therapies can improve the efficacy of CD33-targeted T-cell immunotherapy.

[0355] Second-generation CD33 with a fully human variable domain sequence PAN Antibodies and derivative therapies. Currently, there are no well-characterized antibodies that recognize the C2 set domain of human CD33. FL Whole ECD or human CD33 ΔE2 Antibodies with this specificity were produced in BALB / c, CD1, and F1 mice injected with an immunogen containing a mouse IgG1 Fc domain linked to the entire ECD. A series of mouse anti-human antibodies CD33 PAN Data on the antibody were recently reported (Godwin et al., Leukemia. 2021, DOI: 10.1038 / s41375-021-01160-1). Because the immunogenicity of the mouse amino acid sequence is a potential clinical issue, a second immunization campaign was conducted to produce an antibody with a fully human variable domain sequence using the same CD33 immunogen in humanized mice. As shown in Figures 6A and 6B, CD33 FL A hybridoma that recognizes only CD33 ΔE2 and CD33 FLJoin to both (i.e., CD33 PAN Antibody specificity) It was identified along with several hybridomas. The binding specificity of these antibodies to human CD33 was confirmed by experiments using CD33+ML-1 cells and ML-1 sublines in which CD33 was removed by CRISPR / Cas9-mediated gene editing. Carterra's summary of biophysical characterization studies is shown in Figures 7A and 7B. For further characterization, 1E6 underwent antibody internalization experiments. As shown in Figure 8, 1E6 actually bound the parental mouse CD33 used in GO. Vセット It was internalized into human AML cells with pharmacokinetics similar to those of the antibody P67.6. Since hybridomas derived from Trianni mice secrete chimeric antibodies (human variable binding sequence, mouse constant region), recombinant fully human CD33 was internalized using species conversion methodology. PAN Antibodies were produced. Figure 9 shows an example (1E6 antibody with a human IgG1 framework).

[0356] Human CD33 PAN In a proof-of-principle study regarding the therapeutic value of the antibody, 1E6 / CD3 BsAb was constructed in scFv-scFv format. Mouse CD33 PAN Similar to what was found regarding CD3 BsAb (Godwin et al., Leukemia. 2021, DOI:10.1038 / s41375-021-01160-1), 1E6 / CD3 BsAb was highly potent against CD33+ human acute leukemia cells (Figure 10) but lacked activity against CD33 knockout cells (Figure 11). 1E6 / CD3 BsAb is CD33 FL and CD33 ΔE2 REH cells that overexpress both also kill CD33 Vセット / CD3 BsAb is CD33 FL It was active only against expressing cells (Figure 12). Finally, it was determined that 1E6 / CD3 BsAb exhibits robust activity in vitro against various primary AML patient samples (Figure 13).

[0357] (xiii) Final paragraph. The nucleic acids and amino acid sequences provided herein are as defined in 37 CFR §1.822 and are indicated using abbreviations for nucleotide bases and amino acid residues as shown in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. Although only one strand of each nucleic acid sequence is shown, it is understood that complementary strands are also included in appropriate embodiments.

[0358] To the extent not expressly provided herein, coding sequences for the proteins disclosed herein and protein sequences for the coding sequences disclosed herein can be readily derived by those skilled in the art.

[0359] The precise amino acid sequence boundaries of a given CDR or FR can be easily determined using one of several well-known schemes, including: Kabat et al. (1991) "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273: pp. 927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262: pp. 732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86: pp. 9268-9272 (AbM numbering scheme); Lefranc MP et al. (2003) Dev Comp Immunol This includes those described in 27(1):55-77 (IMGT numbering scheme) and Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions corresponding to insertion letters, e.g., "30a", and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. In certain embodiments, the antibody CDR sequences disclosed herein follow Kabat numbering.

[0360] This includes variants of sequences disclosed and referenced herein. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR® (Madison, Wisconsin) software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the relevant amino acid families in their side chains.

[0361] Antibody variants may include those having one or more conserved amino acid substitutions or one or more non-conservative substitutions that do not adversely affect protein binding.

[0362] In a particular embodiment, V L The region is disclosed V L V which may originate from or is obtained based on and disclosed L Compared to the above, the modified V may include one or more insertions (e.g., two, three, four, five, six, seven, eight, nine, ten), one or more deletions (e.g., two, three, four, five, six, seven, eight, nine, ten), one or more amino acid substitutions (e.g., conservative amino acid substitutions), or combinations of the changes mentioned above. Insertions, deletions, or substitutions may result in each CDR containing no changes or containing one, two, or three or fewer changes, and being modified V L Provided that an antibody containing this region can still specifically bind to its target epitope with the same affinity as the wild-type binding domain, the V region contains the amino-terminus, carboxyl-terminus, or both of these ends. L It can exist anywhere within the domain.

[0363] In a particular embodiment, V H The region is disclosed V H V which may be derived from or obtained based thereon, as disclosed herein HCompared to the above, the modified V may include one or more insertions (e.g., two, three, four, five, six, seven, eight, nine, ten), one or more deletions (e.g., two, three, four, five, six, seven, eight, nine, ten), one or more amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or combinations of the changes mentioned above. Insertions, deletions, or substitutions may result in each CDR containing no changes or containing one, two, or three or fewer changes, and being modified V H Provided that an antibody containing this region can still specifically bind to its target epitope with the same affinity as the wild-type binding domain, the V region contains the amino-terminus, carboxyl-terminus, or both of these ends. H It can exist anywhere within the domain.

[0364] In certain embodiments, conservative amino acid substitutions may not substantially alter the structural features of the reference sequence (for example, the substituted amino acid does not tend to cleave the helix that would occur within the reference sequence or disrupt other types of secondary structures that characterize the reference sequence). Examples of secondary and tertiary structures of polypeptides recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature, 354:105 (1991).

[0365] Appropriate conservation substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th edition, 1987, The Benjamin / Cummings Pub.Co., p. 224). Naturally occurring amino acids are generally classified into the following conserved substitution families: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (Acidic): Aspartic acid (Asp) and Glutamic acid (Glu); Group 3: (Acidic; also classified as polar negative-charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (Basic; also classified as polar positive-charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6: (Large aliphatic, nonpolar residues): Isoleucine (Ile) Group 7 (non-charged polar): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, non-polar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0366] When making such modifications, the hydroxyl index of amino acids may be considered. The importance of the hydroxyl amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, J.Mol.Biol.157(1), pp. 105-132). Each amino acid is assigned a hydroxyl index based on its hydrophobic and charge characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: Ile (+4.5), Val (+4.2), Leu (+3.8), Phe (+2.8), Cys (+2.5), Met (+1.9), Ala (+1.8), Gly (-0.4), Thr (-0.7), Ser (-0.8), Trp (-0.9), Tyr (-1.3), Pro (-1.6), His (-3.2), Glutamate (-3.5), Gln (-3.5), Aspartic acid (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).

[0367] In the art, it is known that by substituting a particular amino acid with another amino acid having a similar hydroxyl index or score, a protein with similar biological activity can still be obtained; that is, a biologically functionally equivalent protein can still be obtained. When such modifications are made, substitutions of amino acids with a hydroxyl index of ±2 are preferred, substitutions of amino acids with a hydroxyl index of ±1 are particularly preferred, and substitutions of amino acids with a hydroxyl index of ±0.5 are even more particularly preferred. In the art, it is also understood that similar amino acid substitutions can be efficiently carried out based on hydrophilicity.

[0368] As detailed in U.S. Patent No. 4,554,101, the following hydrophilic values ​​are assigned to amino acid residues: Arg (+3.0), Lys (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), Ser (+0.3), Asn (+0.2), Gln (+0.2), Gly (0), Thr (-0.4), Pro (-0.5±1), Ala (-0.5), His (-0.5), Cys (-1.0), Met (-1.3), Val (-1.5), Leu (-1.8), Ile (-1.8), Tyr (-2.3), Phe (-2.5), Trp (-3.4). It is understood that by substituting an amino acid with another amino acid having a similar hydrophilicity, it is still possible to obtain a biologically equivalent protein, specifically an immunologically equivalent protein. In such modifications, substitutions of amino acids with a hydrophilicity of ±2 are preferred, substitutions of amino acids with a hydrophilicity of ±1 are particularly preferred, and substitutions of amino acids with a hydrophilicity of ±0.5 are even more particularly preferred.

[0369] As outlined above, amino acid substitutions may be based on the related similarities of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size.

[0370] As shown elsewhere, gene sequence variants may include codon optimization variants, sequence polymorphisms, splice variants, and / or mutations that do not statistically significantly affect the function of the coding protein.

[0371] Variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity with respect to the proteins, nucleic acids, or gene sequences disclosed herein.

[0372] In certain embodiments, the variant comprises or is a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with respect to the antibody sequences disclosed herein. In certain embodiments, the variant comprises a light chain variable region (V L ) and / or heavy chain variable region (V H ) or both, containing or comprising sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% sequence identity, wherein each CDR contains no changes or contains one, two, or three or fewer changes from a reference antibody or fragment or derivative thereof disclosed herein, which specifically binds to the C2 set Ig-like CD33 epitope regardless of the presence or absence of the V set Ig-like domain, or binds to the V set Ig-like domain according to the epitope specificity of the antibody described herein.

[0373] "Sequence identity %" refers to the degree of association between two or more sequences determined by comparing those sequences. In the art, "identity" also means the degree of sequence association between such sequences, determined by the correspondence between protein sequences, the correspondence between nucleic acid sequences, or the correspondence between gene sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Develeux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to maximize the agreement between the tested sequences. Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity percentage calculation can be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignment can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, pp. 151-153 (1989)) with default parameters (gap penalty = 10, gap length penalty = 10).Related programs include the GCG program suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J.Mol.Biol.215: pp. 403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992, pp. 111-120, Editor: Suhai, Sandor, Publisher: Plenum, New). This also includes York, NY. In the context of this disclosure, it is understood that when sequence analysis software is used for the analysis, the results of the analysis are based on the “default values” of the program mentioned. As used herein, “default values” means any set of values ​​or parameters that are initially set in the software during initial initialization.

[0374] The variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions, giving them the same function as the reference sequences. Examples of stringent hybridization conditions include incubation overnight in a 42°C solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured fragmented salmon sperm DNA, followed by washing the filter in 0.1×SSC at 50°C. Modifications of the stringency of hybridization and signal detection are primarily achieved by manipulating the formamide concentration (lowering the percentage of formamide reduces stringency), salt conditions, or temperature. For example, moderately high stringency conditions include overnight incubation in a 37°C solution containing 6×SSPE (20×SSPE = 3M NaCl, 0.2M NaH2PO4, 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA, followed by washing at 50°C with 1×SSPE and 0.1% SDS. To further reduce stringency, the washing performed after stringent hybridization may be carried out with a higher salt concentration (e.g., 5×SSC). Variations of the above conditions can be achieved by including and / or replacing them with alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhart's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of specific blocking reagents may necessitate modifications to the hybridization conditions described above due to compatibility issues.

[0375] "Specifically binding" means that the binding domain (e.g., a bispecific antibody binding domain or a ligand that targets nanoparticle-selective cells) and its corresponding binding molecule do not significantly associate with any other molecules or components in the relevant environmental sample. 5 M -1 The above affinity or K a This refers to association at a specific binding interaction equilibrium binding constant having units of 1 / M. Binding domains can be classified as "high affinity" or "low affinity." In certain embodiments, a "high affinity" binding domain is defined as K a at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 This refers to a binding domain that is K. In a particular embodiment, the "low affinity" binding domain is K a up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 It refers to the binding domain. Alternatively, affinity is the equilibrium dissociation constant (K) of a specific binding interaction having units of M. d )(For example, 10 -5 M~10 -13 It can be defined as M). In certain embodiments, the binding domain may be “affinity enhanced,” which means that the binding domain is selected or manipulated so that it binds more strongly to the corresponding binding molecule than the wild-type (or parent) binding domain. For example, affinity enhancement is achieved by increasing the K binding domain to the corresponding binding molecule than the reference binding domain. a This may be due to a large (equilibrium association constant), or the K for the corresponding binding molecule is greater than that of the reference binding domain. dThis may be due to a small (dissociation constant), or the off-rate (K) relative to the corresponding binding molecule is lower than that of the reference binding domain. off This may be due to a low binding affinity. Various assays are known to detect binding domains that specifically bind to specific corresponding binding molecules and to determine binding affinity, such as Western blotting, ELISA, and BIACORE® analysis (see, for example, Scatchard et al., 1949, Ann. NYAcad. Sci. 51:660; and U.S. Patents 5,283,173, 5,468,614, or equivalent literature).

[0376] Unless otherwise specified, the implementation of this disclosure may utilize conventional techniques in immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989); FMAusubel et al., eds., Current Protocols in Molecular Biology, (1987); the series Methods in Enzymology (Academic Press, Inc.); M. MacPherson et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RIFreshney, ed. Animal Cell Culture (1987).

[0377] As will be understood by those skilled in the art, each embodiment disclosed herein includes, is essentially, or may include the specific elements, processes, components, or constituents described herein. Therefore, the terms “include” or “including” should be interpreted as “comprise,” “consist of,” or “essentially consist of.” The transitional terms “comprise” or “comprises” mean to include, but not limited to, unspecified elements, processes, components, or constituents, even if they are the main component, and that it may include them. The transitional phrase “consist of” excludes any unspecified elements, processes, components, or constituents. The transitional phrase “essentially consist of” limits the scope of the embodiment to specific elements, processes, components, or constituents and those that do not substantially affect the embodiment. Substantial effect would result in a statistically significant reduction in antibody-antigen binding.

[0378] Unless otherwise specified, all numbers used in this specification and the claims to represent component amounts, properties, such as molecular weight and reaction conditions, shall be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise specified, the numerical parameters shown in this specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At the very least, without intending to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted by applying common rounding techniques, taking into account the number of significant figures reported. Where further clarification is required, the term “approximately” when used with a given number or range has a meaning that will be reasonably understood by those skilled in the art, namely, that is, that it is somewhat more or somewhat less than the given value or range, such as within ±20% of the given value, within ±19% of the given value, within ±18% of the given value, within ±17% of the given value, within ±16% of the given value, within ±15% of the given value, within ±14% of the given value, within ±13% of the given value, within ±12% of the given value, within ±11% of the given value, within ±10% of the given value, within ±9% of the given value, within ±8% of the given value, within ±7% of the given value, within ±6% of the given value, within ±5% of the given value, within ±4% of the given value, within ±3% of the given value, within ±2% of the given value, or within ±1% of the given value.

[0379] Regardless of whether the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. However, each numerical value inherently contains a certain degree of error that inevitably results from the standard deviation observed in its respective test measurement.

[0380] In the context describing the present invention (in particular in the context of the following claims), the terms “one (a),” “one (an),” “it,” and similar reference subjects shall be construed to include both singular and plural unless otherwise specified herein or unless the context clearly contradicts it. The descriptions of ranges of values ​​herein are merely intended as a way to simplify the reference to each individual value that falls within such range. Unless otherwise specified herein, each individual value is incorporated herein as it would be if it were described separately herein. Unless otherwise specified herein or unless the context clearly contradicts it otherwise, all methods described herein may be carried out in any suitable order. The use of any or all of the example or illustrative words provided herein (e.g., “etc.”) is merely intended to facilitate the understanding of the present invention and does not limit the scope of the present invention as claimed without such words. No word herein should be construed as indicating any non-claiming element essential to the practice of the present invention.

[0381] Grouping alternative elements or embodiments of the Invention disclosed herein shall not be construed as limitation. Each group member may be referred to and claimed individually, or in any combination with members of other groups or other elements found herein. For convenience and / or patentability reasons, it is anticipated that one or more members of a group may be included in or excluded from a group. In the event of any such inclusion or exclusion, this specification shall be deemed to include the modified group and thus satisfy all descriptions of the Markush group used in the appended claims.

[0382] This specification describes certain embodiments of the Invention, including those that the inventors recognize as being the best mode for carrying out the Invention. Naturally, variations of these described embodiments will become apparent to those skilled in the art by reading the foregoing description. The inventors anticipate that those skilled in the art will utilize such variations as needed, and the inventors intend to carry out the Invention in a manner different from those specifically described herein. Accordingly, the Invention includes all variations and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise specified herein, or unless otherwise clearly contradictory to the context, any combination of the above elements is included in the Invention in all possible variations.

[0383] Furthermore, numerous references are made throughout this specification to patents, printed publications, scholarly articles, and other documents (referenced herein). Each of these references is individually incorporated into this specification by reference, with respect to its reference teachings in their entirety.

[0384] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other possible modifications are also within the scope of the present invention. Therefore, alternative configurations of the present invention may be used, for example, but not limited to, in accordance with the teachings herein. Accordingly, the present invention is not limited to those expressly presented and described herein.

[0385] Details provided herein are illustrative and are intended only to illustrate preferred embodiments of the invention, and are presented to provide what is considered most useful and easily understandable as an explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, there is no intention to provide structural details of the invention in more detail than is necessary for a basic understanding of the invention, but rather to make it clear to those skilled in the art, by using the explanation in conjunction with the drawings and / or examples, how some forms of the invention can be embodied in practice.

[0386] The definitions and descriptions used in this disclosure are intended and will prevail in any future interpretation unless explicitly and unambiguously modified in the following examples, or if applying the meaning would render any interpretation meaningless or essentially meaningless. Where applying the interpretation of a term would render it meaningless or essentially meaningless, the definition shall be taken from Webster's Dictionary, Third Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. (i) CDRL1 having the sequence described in Sequence ID No. 24, CDRL2 having the sequence described in Sequence ID No. 25, CDRL3 having the sequence described in Sequence ID No. 26, CDRH1 having the sequence described in Sequence ID No. 27, CDRH2 having the sequence described in Sequence ID No. 28, and CDRH3 having the sequence described in Sequence ID No. 29, (ii) A CDR having the sequence described in Sequence ID No. 183, CDRL2 having sequence AAS, CDRL3 having the sequence described in Sequence ID No. 26, CDRH1 having the sequence described in Sequence ID No. 184, CDRH2 having the sequence described in Sequence ID No. 185, and CDRH3 having the sequence described in Sequence ID No. 29, (iii) CDRL1 having the sequence described in Sequence ID No. 24, CDRL2 having the sequence described in Sequence ID No. 204, CDRL3 having the sequence described in Sequence ID No. 26, CDRH1 having the sequence described in Sequence ID No. 205, CDRH2 having the sequence described in Sequence ID No. 206, and CDRH3 having the sequence described in Sequence ID No. 207, or (iv) CDRL1 having the sequence described in Sequence ID No. 24, CDRL2 having the sequence described in Sequence ID No. 204, CDRL3 having the sequence described in Sequence ID No. 26, CDRH1 having the sequence described in Sequence ID No. 222, CDRH2 having the sequence described in Sequence ID No. 223, and CDRH3 having the sequence described in Sequence ID No. 207 An antibody or its antigen-binding fragment comprising a set of complementarity-determining regions (CDRs) having [a specific characteristic].

2. A variable light chain having the sequence described in Sequence ID No. 55 and a variable heavy chain having the sequence described in Sequence ID No. 56, or Variable light chain having at least 90% sequence identity with the sequence described in Sequence ID No. 55 and variable heavy chain having at least 90% sequence identity with the sequence described in Sequence ID No. 56 The antibody or antigen-binding fragment thereof according to claim 1, having the above characteristics.

3. The antibody or antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is a single-stranded variable fragment (scFv) that is VH-VL oriented or VL-VH oriented.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein scFv has the sequence described in SEQ ID NO: 234 or 235.

5. The antibody or antigen-binding fragment thereof according to claim 1, as part of a bispecific antibody having a second binding domain that binds to CD3.

6. The second binding domain is A variable light chain having the sequence described in Sequence ID No. 162 and a variable heavy chain having the sequence described in Sequence ID No. 161, or Variable light chain having at least 90% sequence identity with the sequence described in Sequence ID No. 162 and variable heavy chain having at least 90% sequence identity with the sequence described in Sequence ID No. 161 The antibody or antigen-binding fragment thereof according to claim 5, having the above characteristics.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein the bispecific antibody has the sequence described in SEQ ID NO: 242 or 243.

8. An antibody or its antigen-binding fragment comprising a set of complementarity-determining regions (CDRs) defined by IMGT, Kabat, North, or Chothia as 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3.

9. CD33 targeting agents comprising a binding domain containing a set of complementarity-determining regions (CDRs) of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 as defined by IMGT, Kabat, North, or Chothia, as part of an anti-CD33 immunotoxin, anti-CD33 antibody-drug conjugate, anti-CD33 antibody-radioisotope conjugate, anti-CD33 bispecific antibody, anti-CD33 bispecific immune cell-inducing antibody, anti-CD33 tripspecific antibody, and / or anti-CD33 quadruspecific antibody.

10. Anti-CD33 immunotoxins, anti-CD33 antibody-drug conjugates, anti-CD33 antibody-radioisotope conjugates, anti-CD33 bispecific antibodies, anti-CD33 bispecific immune cell-inducing antibodies, anti-CD33 trispecific antibodies, and / or as part of anti-CD33 quadrispecific antibodies. Variable light and heavy chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3; or Sequences having at least 90% sequence identity to the variable light chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3, and sequences having at least 90% sequence identity to the corresponding variable heavy chains of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 A CD33 targeting agent containing a binding domain.

11. The CD33 targeting agent according to claim 9 or 10, wherein the CD33 targeting agent comprises an anti-CD33 immunotoxin, and the toxin comprises a holotoxin or a hemitoxin.

12. The CD33 targeting agent according to claim 9 or 10, wherein the CD33 targeting agent comprises an anti-CD33 immunotoxin, the toxin comprising abrin, bougain, bryodin 1, diphtheria toxin (DT), geronin, pygmyxin lectin, modesin, pokeweed antiviral protein (PAP), Pseudomonas exotoxin (PE), lysine and / or saporin.

13. The CD33 targeting agent contains an anti-CD33 antibody-drug conjugate, and the drugs include monomethyl auristatin E [MMAE], vedotin, drastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin, PNU-159682, anthracycline, duocalmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, erinafid, taxol, cytochalasin B, and gramin. A CD33 targeting agent according to claim 9 or 10, comprising cidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, and / or propranolol.

14. The CD33 targeting agent according to claim 9 or 10, wherein the CD33 targeting agent comprises an anti-CD33 antibody-radioisotope conjugate, the radioisotopes comprising arsenic-72, arsenic-74, iodine-131, indium-111, yttrium-90, lutetium-177, astatine-211, actinium-225, bismuth-212 and / or bismuth-213.

15. The CD33 targeting agent comprises an anti-CD33 antibody-radioisotope conjugate, and the radioisotope is 225Ac, 228Ac, １１１ Ag, １２４ Am, ７４ As, ２１１ At, ２０９ At, １９４ Au, １２８ Ba, ７ Be, ２０６ Bi, ２４５ Bk, ２４６ Bk, ７６ Br, １１ C, ４７ Ca, ２５４ Cf, ２４２ Cm, ５１ Cr, ６７ Cu, １５３ Dy, １５７ Dy, １５９ Dy, １６５ Dy, １６６ Dy, １７１ Er, ２５０ Es, ２５４ Es, １４７ Eu, １５７ Eu, ５２ Fe, ５９ Fe, ２５１ Fm, ２５２ Fm, ２５３ Fm, ６６ Ga, ７２ Ga, １４６ Gd, １５３ Gd, ６８ Ge, １７０ Hf, １７１ Hf, １９３ Hg, １９３ mHg, １６０ mHo, １３０ I, １３１ I, １３５ I, １１４ mIn, １８５ Ir, ４２ K, ４３ K, ７６ Kr, ７９ Kr, ８１ mKr, １３２ La, ２６２ Lr, １６９ Lu, １７４ mLu, １７６ mLu, ２５７ Md ２６０ Md ２８ Mg ５２ Mn ９０ Mo ２４ Na ９５ Nb １３８ Nd ５７ Ni ６６ Ni ２３４ Np １５ O １８２ Os １８９ mOs １９１ Os ３２ P ２０１ Pb １０１ Pd １４３ Pr １９１ Pt ２４３ Pu ２２５ Ra ８１ Rb １８８ Re １０５ Rh ２１１ Rn １０３ Ru ３５ S ４４ Sc ７２ Se １５３ Sm １２５ Sn ９１ Sr １７３ Ta １５４ Tb １２７ Te ２３４ Th ４５ Ti １６６ Tm ２３０ U ２３７ U ２４０ U<构 ４８ V １７８ W １８１ W １８８ W １２５ Xe １２７ Xe １３３ Xe １３３ mXe １３５ Xe ８５ mY ８６ Y ９０ Y ９３ Y １６９ Yb １７５ Yb ６５ Zn ７１ mZn ８６ Zr ９５ Zr and / or ９７ The CD33 targeting agent according to claim 9 or 10, comprising Zr. It should be noted that there seems to be an incorrect tag "<构 ４８ " in the original text. It might be a formatting or encoding issue. If this is a critical error, it may need to be corrected in the source before accurate translation and further processing.

16. A CD33 targeting agent according to claim 9 or 10, comprising a multispecific antibody.

17. The CD33 targeting agent according to claim 16, wherein the multispecific antibody comprises a bispecific antibody, a triplicate antibody, or a quadruplicate antibody.

18. The CD33 targeting agent according to claim 16, wherein the multispecific antibody includes a binding domain that activates immune cells.

19. The CD33 targeting agent according to claim 18, wherein the immune cells are T cells, natural killer (NK) cells, or macrophages.

20. The CD33 targeting agent according to claim 19, wherein the T cells are CD3 T cells, CD4 T cells, CD8 T cells, central memory T cells, effector memory T cells and / or naive T cells.

21. A CD33 targeting agent according to claim 18, wherein the binding domain that activates immune cells binds to CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Rα, or MARCO.

22. A CD33 targeting agent according to claim 18, having the sequence described in any one of sequence numbers 157 or 238-245.

23. The CD33 targeting agent according to claim 18, wherein the binding domain activates T cells and comprises a CDR of OKT3 antibody, a CDR of 4B4-D7 antibody, a CDR of 4E7-C9 antibody, a CDR of 18F5-H10 antibody, a CDR of TGN1412 antibody, a CDR of OKT8 antibody, a variable light chain having the sequence described in SEQ ID NO: 162, and a variable heavy chain having the sequence described in SEQ ID NO: 161, or a TCR.

24. Fv, Fab, Fab', F(ab') of 1H10, 1A9, 1E6, 1D2, 1B9, 1H8, 2D3, or 2E3 ２ A CD33 targeting agent according to claim 9 or 10, comprising, or a single-stranded Fv fragment (scFv).

25. The CD33 targeting agent according to claim 24, wherein scFv has the sequence described in any one of sequence numbers 230 to 237.

26. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a linker, or the CD33 targeting agent according to claim 9 or 10.

27. The CD33 targeting agent according to claim 26, wherein the linker is a Gly-Ser linker.

28. Gly-Ser linker, (Gly ｘ Ser ｙ ) ｎ The CD33 targeting agent according to claim 27, comprising, where x and y are independently integers from 0 to 10, provided that both x and y are not 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

29. Gly-Ser linker, (Gly ４ Ser ）４ (Sequence ID 125), (Gly ４ Ser) ３ (Sequence No. 126), (Gly ４ Ser) ２ (Sequence No. 127), (Gly ４ Ser) １ (Sequence No. 128), (Gly ３ Ser) ２ (Sequence No. 129), (Gly ３ Ser) １ (Sequence ID 130), (Gly ２ Ser) ２ (Sequence ID 131) or (Gly ２ Ser) １ A CD33 targeting agent according to claim 28, comprising GGSGGGGGGSG (SEQ ID NO: 132), GGSGGGGSGSG (SEQ ID NO: 133), or GGSGGGGSG (SEQ ID NO: 134).

30. The antibody or antigen-binding fragment according to claim 8, wherein the antibody or antigen-binding fragment thereof is PEG-modified.

31. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody comprises an Fc modification.

32. The antibody or antigen-binding fragment thereof according to claim 31, wherein the Fc modification comprises the M428L / N434S, G236A / S239D / A330L / I332E (GASDALIE), huIgG4 ProAlaAla, huIgG2m4, and / or huIgG2sigma mutation.

33. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or 8, and / or the CD33 targeting agent according to claim 9 or 10, formulated for administration to a target.

34. Cells genetically modified to express the antibody or antigen-binding fragment thereof described in claim 8 and / or the CD33 targeting agent described in claim 9 or 10, formulated for administration to a target.

35. The cells according to claim 34, which are in vivo or ex vivo.

36. The cell according to claim 34, which is a T cell, a B cell, a natural killer (NK) cell, a monocyte / macrophage, a hematopoietic stem cell (HSC), or a hematopoietic progenitor cell (HPC).

37. The cell according to claim 34, which is a T cell selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells.

38. The cell according to claim 34, wherein the cell is a CD8+ T cell.

39. A formulation comprising the cell population described in claim 34 and a pharmaceutically acceptable carrier.

40. A method for treating a CD33-related disorder in a subject requiring the treatment thereof, comprising administering a therapeutically effective amount of the composition and / or the formulation according to claim 39 to the subject, thereby treating a CD33-related disorder in the subject requiring the treatment thereof.

41. The method according to claim 40, wherein the CD33-related disorder includes acute myeloid leukemia (AML).

42. The method according to claim 40, wherein the CD33-related disorder includes acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), mast cell leukemia, myelodysplastic syndrome (MDS), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or megakaryocytic leukemia.

43. The method according to any one of claims 40, wherein the population of cells in the formulation is autologous or homogeneous to the subject.

44. To determine whether the subject expresses or lacks the V-set domain of CD33, and If the target organism expresses the V-set domain of CD33, One or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9, and One or more binding domains from 1H8, 2D3, and 2E3 The method according to any one of claims 40, further comprising selecting a combination therapy comprising a composition comprising the above.

45. To determine whether the subject expresses or lacks the V-set domain of CD33, and If the subject does not express the V-set domain of CD33, A composition comprising one or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9. The method according to any one of claims 40, further comprising selecting a therapy that includes the following:

46. A method for activating an immune response against CD33-expressing cells in a subject requiring the same, comprising administering a therapeutically effective amount of the composition according to claim 33 and / or the formulation according to claim 39 to the subject, thereby activating an immune response against CD33-expressing cells in a subject requiring the same.

47. The method according to claim 46, wherein the CD33-expressing cells include acute myeloid leukemia (AML) cells.

48. The method according to claim 46, wherein the CD33-expressing cells include cells of acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), mast cell leukemia, myelodysplastic syndrome (MDS), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or megakaryocytic leukemia.

49. The method according to any one of claims 46, wherein the population of cells in the formulation is autologous or homogeneous to the subject.

50. To determine whether the subject expresses or lacks the V-set domain of CD33, and If the target organism expresses the V-set domain of CD33, One or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9, and A composition comprising one or more binding domains from among 1H8, 2D3, and 2E3. The method according to any one of claim 46, further comprising selecting a combination therapy including the following:

51. To determine whether the subject expresses or lacks the V-set domain of CD33, and If the subject does not express the V-set domain of CD33, A composition comprising one or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9. The method according to any one of claim 46, further comprising selecting a therapy that includes the following:

52. A kit comprising a composition containing one or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9, and one or more binding domains from among 1H8, 2D3, and 2E3.

53. A kit comprising a composition containing one or more binding domains from among 1H10, 1A9, 1E6, 1D2, and 1B9, and a composition containing one or more binding domains from among 1H8, 2D3, and 2E3.

Images