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  • A61K47/6853—Carcino-embryonic antigens
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• • C—CHEMISTRY; METALLURGY
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Definitions

• This document provides methods and materials involved in binding a molecule (e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC) to a CD66e polypeptide.
• a molecule e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC
• binders e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, or ADCs
• This document also provides cells (e.g., host cells) designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to a CD66e polypeptide and methods and materials for using such cells to treat cancer.
• binders e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers
• the checkpoint inhibitor can be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP- 224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA- 170, BMS-986189, and ipilimumab.
• the number of cancer cells within the mammal can be reduced following the administering steps (a) and (b).
• the light chain variable domain or region can comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32.
• the antibody can be a monoclonal antibody.
• the antibody can be an scFv antibody.
• the antigen binding fragment can comprise (or consist essentially of or consist of): (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:l (or SEQ ID NO:l with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ IDNO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions
• Figure 5 depicts the structure of an exemplary Ig and provides the amino acid and nucleic acid sequences of an exemplary hinge, CH2, and CH3 regions/domains.
• Figures 9A and 9B depict the amino acid sequences of an exemplary heavy chain variable domain (Figure 9A) and an exemplary light chain variable domain (Figure 9B) of an exemplary scFv.
• the CDRs and framework sequences of each also are delineated.
• An exemplary linker amino acid sequence such as a linker amino acid sequence set forth in Figure 10 can be used to link the heavy chain variable domain and the light chain variable domain together to form a scFv.
• Figure 15 depicts the amino acid sequences of exemplary intracellular signaling domains that can be used to design a CAR.
• Figure 25 Binding of 1G9 Fab to different domains of CEACAM family members in flow-cytometry analysis.
• Figure 40 Fluorescence intensity histogram of the remained antibody levels (1G9 and 1C1 hlgGl) on NCI-H660 cell surface after the indicated incubation times at 37 °C and the quantification of mean fluorescence intensity (MFI) for 1G9 and 1C1 hlgGl.
• Figure 41 Design of scFv of 1G9, and the binding of scFv of 1G9 in different VH/VL orientations and in use of linkers to A3B3 domain of CEACAM5.
• Figure 44 Cytotoxic activity of anti-CEACAM5 CAR-T against CEACAM5- positive cell line (NCI-H660), stably CEACAM5-expressing Dul45 cell line (Dul45- CEACAM5), and CE AC AM5 -negative cell line (Dul45).
• Figure 48 Tumor growth curves and body weight of individual mice for the indicated hlgGl treatment groups in Dul45-CEACAM5 and Dul45 tumors.
• antibody as used herein includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multi-specific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, single-chain variable fragment antibodies (e.g., scFv antibodies), and tandem single-chain variable fragments antibody (e.g., taFv).
• Adiabody can include two chains, each having a heavy chain variable domain and a light chain variable domain, either from the same or from different antibodies (see, e.g., Hornig and Farber- Schwarz, Methods Mol. Biol ., 907:713-27 (2012); and Brinkmann and Kontermann, MAbs .,
• An anti-CD66e antibody, anti-CD66e antigen binding fragment, or anti-CD66e antibody domain provided herein can be of the IgA-, IgD-, IgE-, IgG-, or IgM-type, including IgG- or IgM-types such as, without limitation, IgGi-, IgG2-, IgG3-, IgG 4 -, IgMi-, and IgM2-types.
• an antibody provided herein e.g., an anti-CD66e antibody
• an antigen binding fragment provided herein e.g., an anti-CD66e antibody fragment
• a CAR provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 12. In some cases, a CAR provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 12 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof.
• a CAR provided herein can be designed to include a hinge of any appropriate length.
• a CAR provided herein can be designed to include a hinge that is from about 3 to about 75 (e.g., from about 3 to about 65, from about 3 to about 50, from about 5 to about 75, from about 10 to about 75, from about 5 to about 50, from about 10 to about 50, from about 10 to about 40, or from about 10 to about 30) amino acid residues in length.
• a linker sequence can be used as a hinge to make a CAR described herein.
• any one of the linker sequences set forth in Figure 10 can be used as a hinge of a CAR described herein.
• a CAR provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13. In some cases, a CAR provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof.
• a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO: 8, followed by a linker such as a linker set forth in Figure 10, followed by a light chain variable domain comprising SEQ ID NO: 16, followed by a hinge such as a hinge/linker set forth in Figure 10 or Figure 13 (e.g., an IgG4-derived hinge, a CD8a hinge, or a linker plus IgG4- derived hinge), followed by a transmembrane domain such as a transmembrane domain set forth in Figure 14 (e.g., a human CD28 transmembrane domain or a CD8a transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Figure 15 (e.g., a human 4-1BB intracellular signaling domain followed by a human E ⁇ 3z intracellular signaling domain).
• a linker such as a link
• a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO: 8, followed by SEQ ID NO: 100, followed by a light chain variable domain comprising SEQ ID NO: 16, followed by SEQ ID NO: 102, followed by SEQ ID NO: 108, followed by SEQ ID NO: 119, followed by SEQ ID NO: 124, followed by SEQ ID NO: 123, followed by SEQ ID NO: 97, followed by SEQ ID NO: 121.
• a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO:24, followed by SEQ ID NO: 100, followed by a light chain variable domain comprising SEQ ID NO:32, followed by SEQ ID NO: 102, followed by SEQ ID NO: 108, followed by SEQ ID NO: 119, followed by SEQ ID NO: 124, followed by SEQ ID NO: 123, followed by SEQ ID NO: 97, followed by SEQ ID NO: 121.
• a cell engager can be designed to include at least one antigen binding domain having the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and at least one other antigen binding domain. That at least one other antigen binding domain can have the ability to bind to any appropriate antigen expressed on the surface of a cell.
• a CD66e polypeptide e.g., a human CD66e polypeptide
• That at least one other antigen binding domain can have the ability to bind to any appropriate antigen expressed on the surface of a cell.
• the cell engager can include an antigen binding domain having the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and one or more (e.g., one, two, or three) antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell.
• a CD66e polypeptide e.g., a human CD66e polypeptide
• one or more (e.g., one, two, or three) antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell.
• a cell engager e.g., a BiTE targeting a CD66e polypeptide
• a cell engager can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, followed by a linker such as a linker set forth in Figure 10, followed by a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker such as a hinge/linker set forth in Figure 10 or Figure 13 (e.g., SEQ ID NO:78), followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).
• an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).
• a cell engager e.g., a BiTE targeting a CD66e polypeptide
• a cell engager can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO:32, followed by a linker such as a linker set forth in Figure 10, followed by a heavy chain variable domain comprising SEQ ID NO:24, followed by a linker such as a hinge/linker set forth in Figure 10 or Figure 13 (e.g., SEQ ID NO:78), followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).
• a linker such as a linker set forth in Figure 10
• a linker such as a hinge/linker set forth in Figure 10 or Figure 13
• an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 s
• a cell engager e.g., a BiTE targeting a CD66e polypeptide
• IgG e.g., IgGl
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a CD66e polypeptide e.g., a human CD66e polypeptide
• an Fab can be designed to include the six CDRs set forth in Figures 2 A and 2B and the framework regions set forth in Figures 2A and 2B except that framework region 1 having the amino acid set forth in SEQ ID NO:4 is replaced with a framework region 1 having the amino acid set forth in SEQ ID NO: 58 or a framework region 1 having the amino acid set forth in SEQ ID NO:67 or a framework region 1 having the amino acid set forth in SEQ ID NO:72.
• a scFv can be designed to include the six CDRs set forth in Figures 2Aand 2B and the framework regions set forth in Figures 2A and 2B.
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a binder can include (a) a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 8 and/or (b) a light chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 16.
• an antibody or antigen binding fragment provided herein can have the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide), can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, and can include a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 16 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the light chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:9, 10, and 11.
• a CD66e polypeptide e.g., a
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a CD66e polypeptide e.g., a human CD66e polypeptide
• a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 3, and/or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 9, (ii) a CDR2 that comprises
• a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:9” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:9, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO: 9, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:9, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide).
• Examples of a CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:9 include, without limitation, those set forth in Table 4.
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a binder having any of the CDRs set forth in Figures 3 A or 3B can be designed to include framework regions as set forth in Figures 3 A and 3B or can be designed to include one or more framework regions from another antibody or antibody fragment.
• an Fab can be designed to include the six CDRs set forth in Figures 3 A and 3B and the framework regions set forth in Figures 3 A and 3B except that framework region 1 having the amino acid set forth in SEQ ID NO:20 is replaced with a framework region 1 having the amino acid set forth in SEQ ID NO: 58 or a framework region 1 having the amino acid set forth in SEQ ID NO: 67 or a framework region 1 having the amino acid set forth in SEQ ID NO:72.
• a scFv can be designed to include the six CDRs set forth in Figures 3 A and 3B and the framework regions set forth in Figures 3 A and 3B.
• a scFv can be designed to include the six CDRs set forth in Figures 3 A and 3B and the framework regions set forth in Figures 3 A and 3B except that framework region 1 having the amino acid set forth in SEQ ID NO:20 is replaced with a framework region 1 having the amino acid set forth in SEQ ID NO: 58, a framework region 1 having the amino acid set forth in SEQ ID NO:67, or a framework region 1 having the amino acid set forth in SEQ ID NO:72.
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a CD66e polypeptide e.g., a human CD66e polypeptide
• a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24
• a light chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32.
• a binder e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC
• a CD66e polypeptide e.g., a human CD66e polypeptide
• a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:17,
• an antibody or antigen binding fragment provided herein can have the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide), can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:24 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:17, 18, and 19, and can include a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:32 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the light chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:25,
• a single chain antibody e.g., a scFv
• the two regions can be directly connected or can be connected using any appropriate linker sequence.
• a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 or SEQ ID NOs: 17- 19 can be directly connected to a light chain variable domain having the CDRs of SEQ ID NOs:9-ll or SEQ ID NOs:25-27, respectively, via a linker sequence.
• linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain to create a scFv include, without limitation, those linkers set forth in Figure 10.
• Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid,
• a binder e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC
• a binder e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC
• substantially pure refers to the binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) as being substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated.
• a pharmaceutical composition provided herein can be formulated to include one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cells designed to express a CAR having the ability to bind to a CD66e polypeptide, one or more cell engagers, and/or one or more ADCs) provided herein in combination with one or more checkpoint inhibitors such as anti-PD-1 antibodies or PD-1 inhibitors (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, or AMP-514), anti-PD-Ll antibodies or PD-Ll inhibitors (e.g., avelumab, durvalumab, atezolizumab
• a pharmaceutical composition provided herein can be formulated to be a solid or semi-solid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein.
• a binder e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC
• a pharmaceutical composition containing a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a titer of the binder being from about 1 x 10 5 to about 1 x 10 12 (e.g., from about 1 x 10 5 to about 1 x 10 10 , from about 1 x 10 5 to about 1 x 10 8 , from about 1 x 10 6 to about 1 x 10 12 , from about 1 x 10 6 to about 1 x 10 12 , from about 1 x 10 8 to about 1 x 10 12 , from about 1 x 10 9 to about 1 x 10 12 , from about 1 x 10 6 to about 1 x 10 11 , or from about 1 x 10 7 to about 1 x 10 10 ).
• a pharmaceutical composition provided herein can be formulated to be a liquid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 2 mg to about 200 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a nucleic acid provided herein per mL.
• a nucleic acid provided herein per mL.
• compositions e.g., a pharmaceutical composition provided herein
• binders e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs
• a nucleic acid, vector, or host cell e.g., CAR + cells
• a mammal having a CD66e + cancer (e.g., a CD66e + lung cancer, a CD66e + prostate cancer, a CD66e + esophageal cancer, a CD66e + stomach cancer, a CD66e + colorectal cancer, a CD66e + liver cancer, a CD66e + vaginal cancer, or a CD66e + cervical cancer)
• a composition e.g., a pharmaceutical composition
• binders e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs
• an effective amount of a composition containing one or more binders e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs
• a nucleic acid, vector, or host cell e.g., CAR + cells
• a pharmaceutical composition provided herein can be an amount that reduces the number of cancer cells within a mammal having cancer without producing significant toxicity to the mammal.
• an effective frequency of administration of a composition containing one or more binders e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs
• a nucleic acid, vector, or host cell e.g., CAR + cells
• a pharmaceutical composition provided herein can be a frequency that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition.
• an effective frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be from about twice daily to about once a year (e.g., from about twice daily to about once a month, from about twice daily to about once a week, from about once daily to about once a month, or from one once daily to about once a week).
• the frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be daily.
• the frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application.
• the severity of the cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective frequency of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein).
• a composition provided herein e.g., a pharmaceutical composition containing one or more binders provided herein.
• an effective duration of administration of a composition containing one or more binders e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs
• a nucleic acid, vector, or host cell e.g., CAR + cells
• a pharmaceutical composition provided herein can be a duration that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition.
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a label e.g., a covalently attached radioactive, enzymatic, colorimetric, or fluorescent label.
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• cell sorting e.g., fluorescence activated cell sorting
• a binder e.g., an antibody, antigen binding fragment, and/or antibody domain
• a mammal e.g., a human
• a mammal e.g., a human
• a mammal can be assessed using a means for detecting the detectable label.
• a mammal can be scanned to evaluate the location(s) of a labelled binder provided herein within the mammal.
• the mammal can be imaged using NMR or other tomographic techniques.
• Example 1 Obtaining binders having the ability to bind to a human CD66e polypeptide
• CD66e contains anN-terminal Ig variable-region-liken (IgV) domain and six Ig constant region-type 2-like (IgC2-like) domains, N-A1-B1-A2-B2-A3-B3 joined to a GPI-anchor to the plasma membrane.
• IgV Ig variable-region-liken
• IgC2-like domains Ig constant region-type 2-like domains
• the membrane-proximal A3B3 domains are found in splice variants of CD66e in numerous cancers so they were considered potent therapeutic epitopes.
• two Fab antibody fragments (Clones: #1 and #2; Figures 2 and 3) were identified.
• Fc-fused soluble CEACAM5 and CEACAM6 domains were designed and applied to a panning process. Isolated Fabs bound A3B3 domains and dominantly bound B3 domain and N-linked glycans at N612 and N650 of CEACAM5 in high affinity and specificity. Moreover, they did not cross reactive to either other domains of CEACAM5 or CEACAM family members.
• the sequences of Clone #1 were used to make an hlgGl that exhibited CEACAM5-dependent cytotoxic activity in ADCC assays with the NEPC cell line, NCI- H660, and the PrAd cell line, Dul45 in the presence of either primary NK cells or PBMCs. Moreover, constructed third generation CARs containing an scFv having the CDRs of Clone #1 were delivered to T cells that were shown to efficiently kill CEACAM5 positive prostate cancer cells while no detectable cytotoxicity was founded in CEACAM5 negative cells. These results demonstrate an immunotherapeutic potential of Clone #1 hlgGl for NEPC treatment with considerably low off-target toxicity.
• Human immunoglobulin 1 Fc region fused CEACAM5 A3B3 (residues 501-682), CEACAM5 A1B1 (residues 145-322), CEACAM5 A2B2 (residues 323-500), and CEACAM6 AB (residues 145-296) were synthesized and then cloned into pSectag2A plasmid (Invitrogen, V90020). Each plasmid DNA was complexed with PEI- Max (Polysciences, 24765-1) and supplied to culture of the Freestyle human embryonic kidney cell-line (Gibco, R79007) for the transient transfection.
• Fc-fused recombinant proteins were purified by affinity chromatography with protein A resin (Captiva, NC0997253). Elution of bound proteins to protein A was eluted by adding 50 mM Glycine buffer pH 3.0, and then storage buffer was changed to phospho-buffered saline pH 7.4 (PBS) by using PD-10 desalting column (GE, 45-000- 148). Protein purity was estimated in either SDS-PAGE or size exclusion chromatography packed with Superdex 200 increase 10/300 GL (GE healthcare, 28990944). The concentration of each proteins was determined by Nano Drop spectrophotometer 2000C (Thermo, ND2000C).
• N-glycosylation mutants of A3B3 domain of CEACAM5 were constructed by site-directed mutagenesis with Q5-site directed mutagenesis kit (NEB, E0554S), and those proteins were expressed and purified in same manner for wild type of CEACAM5.
• a combinatorial phage-displayed human Fab library (1 c 10 11 clones) was constructed by grafting naturally occurring V(D)J recombination regions of heavy chain (HC) and VJ recombination regions of light chain (LC) into the IGHV3-11 and IGKV1- 39 germline framework, respectively.
• CDR3 complementary determining region 3
• J genes for HC or LC RNA extracted from PBMCs of 50 healthy blood donors was used for cDNA synthesis with SuperscriptTM IV first-strand Synthesis System (Invitrogen, 18091050) and random hexamer and oligo dT primers were applied to annealing step.
• phages were pre-blocked with 3% bovine serum albumin (BSA) in PBS (w/v) for 1 hour at room temperature. Blocked phages incubated with 10 nM biotinylated CEACAM5 A3B3-Fc for 1 hour at room temperature in the presence of 300 nM competitors comprising CEACAM5 AlBl-Fc, CEACAM5 A2B2-Fc, and CEACAM6 AB-Fc.
• BSA bovine serum albumin
• Bound phages were separated by streptavidin coated magnetic beads (Invitrogen, 11-205-D) and washed 10 times with 1 mL of PBS pH 7.4 containing 0.1% Tween-20 (w/v). Elution of bound phages was conducted by adding 10 mM tris-HCl pH 8.0 containing 25 mM dithiothreitol (DTT) for 10 minutes. After three rounds of panning, binding of 192 individual clones was analyzed in ELISA and then selected clones were sequenced after plasmid rescue.
• DTT dithiothreitol
• coli cells were harvested and resuspended in 1/10 volume of periplasm extraction buffer containing polymyxin B (0.5 mg/mL in PBS pH 7.4) and then incubated on ice for an hour. Supernatant was collected then loaded into pre-packed Ni- NTA resin. Bound scFv or Fab was eluted by adding 300 mM imidazole in PBS pH 7.4 and then imidazole was removed.
• IgG preparation IgG cloned plasmid DNA was transfected to HEK239F cells, and expressed for 5-7 days post-transfection. IgGs were purified as previously described in Fc-fused antigen preparation.
• N-linked glycans in A3B3 domains of CEACAM5 may be involved in the binding of 1G9 Fab either directly or indirectly because the A3B3 domains have eight N-linked glycosylation motifs (N-X-S/T; X is natural 19 amino acids except for proline) at N508, N529, N553, N560, N580 in A3 domain and N612, N650, N665 in B3 domain.
• Those N-linked glycans may cover exposed surface of A3B3 domains.
• N580Q mutation slightly improved the binding of 1G9 Fab (Figure 29). These results were corresponded to an image obtained in the electron microscopy (EM) with the complex of 1G9 hlgG and A3B3-Fc ( Figure 33).
• EM electron microscopy
• Figure 33 To visualize location and orientation of N-linked glycans of CEACAM5 and CEACAM6, molecular structures were modeled with added N-linked glycans to the modeling structure. Those four domains showed very similar tertiary structure as expected because they shared over 70% primary sequences and were already known as having Ig constant region-type 2-like (IgC2-like) domains.
• Example 2 In vitro pre-clinical efficacy assessment for hlgGl and CAR-T Cell lines
• pan T cells (Precision for medicine) were activated by Dynabeads human T-activator CD3/CD28 (Gibco), were transduced with lentiviral supernatants with 8 pg/mL polybrene (Sigma), followed by centrifugation for 45 minutes at 800xg (no acceleration and no deceleration), and then incubated at 37°C. 24 hours later, media with viruses was changed, and T cells were expanded in the T cell media (RPMI1640 supplemented with an extra 2 mM glutamax, 10% human serum, and 1% P/S) in the presence of hIL-2 (fed every 2 days, 50 IU/mL, Miltenyi Biotec).
• RPMI1640 supplemented with an extra 2 mM glutamax, 10% human serum, and 1% P/S
• Anti-CEACAM5 CAR-T shows strong cytotoxicity against CEACAM5 -positive prostate cancer cells
• the expression level of 3 rd generation CAR in transduced T cells was 84%, including 30% in CD4 + T cells and 54% in CD8 + T cells, which was similar to the expression level of 2 nd generation CAR, which totaled at 78% (Figure 43).
• IPTG isopropyl b-D-l- thiogalactopyranoside
• a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or SEQ ID NO:27 with one, two, or three amino acid additions, deletions, or substitutions).
• Embodiment 7 The antibody of any one of embodiments 1-2, wherein said antibody comprises said heavy chain variable domain or region of said (ii).
• Embodiment 8 The antibody of embodiment 7, wherein said heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24.
• a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions); or
• a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or SEQ ID NO:27 with one, two, or three amino acid additions, deletions, or substitutions).
• Embodiment 15 The antigen binding fragment of any one of embodiments 11-12, wherein said antigen binding fragment comprises said light chain variable domain or region of said (i).
• Embodiment 18 The antigen binding fragment of embodiment 17, wherein said heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24.
• Embodiment 19 The antigen binding fragment of any one of embodiments 11-12, wherein said antigen binding fragment comprises said light chain variable domain or region of said (ii).
• Embodiment 42 The cell engager of any one of embodiments 32-41, wherein said cell engager comprises a third antigen binding domain.

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• 2022
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