Method for quantifying polypeptide

Abstract

The present application provides a method for quantifying the amount of a polypeptide comprising a portion of an antibody present in a sample (e.g., a plasma or serum sample) wherein the antibody comprises a constant region (e.g., a heavy or light chain constant region) comprising engineered mutations.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of January 11, 2019, application number 201980016834.2 (international application number PCT / US2019 / 013373) and name “Method for Quantifying Polypeptides”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 617,080, filed on January 12, 2018, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence listing submitted as ASCII text file

[0005] The following content submitted as an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 183952028240SEQLIST.txt, Record Date: January 11, 2019, Size: 29KB). Technical Field

[0006] The present invention provides methods for quantifying the amount of a polypeptide comprising an antibody constant region (or a portion thereof) having an engineered mutation in a sample. Background Art

[0007] Proteins, such as antibodies, represent a growing class of therapeutic agents. During preclinical development, candidate therapeutic proteins are extensively analyzed in animal models to assess their pharmacokinetic (PK), pharmacodynamic (PD) and toxicokinetic (TK) properties, evaluate their safety profile, and determine a safe dose for the first human study. During clinical development, the PK, PD and TK properties of therapeutic proteins are further analyzed in human subjects. Data from human studies are used to evaluate the safety and efficacy of therapeutic proteins, establish dosing regimens and / or adjust doses in patient subpopulations. Therefore, it is critical that the methods used to quantify therapeutic proteins in preclinical and clinical samples must be reliable and sensitive.

[0008] Ligand binding assays (LBAs) have traditionally been used to quantify therapeutic proteins due to their high sensitivity, low cost, and high throughput. Despite these advantages, LBAs have limited linear dynamic range, risk of cross-reactivity across metabolites / analogs, and are difficult to multiplex. In addition, developing new antibodies in LBAs for new therapeutic proteins is both expensive and labor-intensive.

[0009] Liquid chromatography tandem mass spectrometry (LC-MS / MS) has become a promising assay platform for quantifying therapeutic proteins. LC-MS / MS assays for bioanalysis of therapeutic proteins in preclinical animal studies typically rely on the quantification of "surrogate peptides" (i.e., peptides whose sequences are unique to the therapeutic protein and not present in the proteome of the preclinical species) as a surrogate measure of the therapeutic protein. Since many therapeutic proteins are derived from human proteins, the sequences of the surrogate peptides may be present in the human proteome, thus hampering accurate quantification of therapeutic proteins in samples obtained from human subjects.

[0010] Therefore, there remains a need in the art for general methods for quantifying therapeutic proteins in preclinical (non-human) and clinical (human) samples. The present disclosure is directed to this need and others.

[0011] All references cited in this application are expressly incorporated herein by reference. Summary of the invention

[0012] Provided is a method for quantifying the amount of a polypeptide comprising a portion of an antibody heavy chain constant region in a sample, the method comprising: (a) digesting a sample comprising a polypeptide comprising a portion of an antibody heavy chain constant region, wherein a portion of the antibody heavy chain constant region comprises an engineered mutation, and wherein digestion produces a peptide fragment derived from the antibody heavy chain constant region, the peptide fragment having a length of 5 to 26 amino acids and comprising the engineered mutation; and (b) analyzing the digested sample by mass spectrometry to determine the amount of the peptide fragment, thereby determining the amount of the polypeptide comprising a portion of an antibody heavy chain constant region in the sample. In certain embodiments according to (or as applied to) any of the above embodiments, the peptide fragment does not comprise methionine (M) or cysteine ​​(C). In certain embodiments according to (or as applied to) any of the above embodiments, the peptide fragment does not comprise asparagine (N) followed by glycine (G) or serine (S). In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises purifying and concentrating the digested sample before mass spectrometry analysis. In certain embodiments according to (or as applied to) any of the above embodiments, the digested sample is purified and concentrated via solid phase extraction (SPE). In certain embodiments according to (or as applied to) any of the embodiments above, the digested sample is purified and concentrated by SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).

[0013] In some embodiments according to (or as applied to) any of the above embodiments, the sample is a whole blood sample, a serum sample, a plasma sample or a tissue sample. In some embodiments according to (or as applied to) any of the above embodiments, the sample is from a mouse, a non-human primate or a human. In some embodiments according to (or as applied to) any of the above embodiments, the non-human primate is a cynomolgus monkey or a rhesus monkey. In some embodiments according to (or as applied to) any of the above embodiments, the sample is digested with at least one enzyme. In some embodiments according to (or as applied to) any of the above embodiments, the at least one enzyme is trypsin, chymotrypsin, glutamyl endopeptidase, lysyl endopeptidase, Asp-N, Arg-C, Glu-C, cyanogen bromide (CnBr) or a combination thereof. In some embodiments according to (or as applied to) any of the above embodiments, the mass spectrum for determining the amount of polypeptide in the sample is liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0014] In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprises a CH1 domain, and wherein the CH1 domain comprises an engineered mutation. In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprises a CH2 domain, and wherein the CH2 domain comprises an engineered mutation. In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprises a CH3 domain, and wherein the CH3 domain comprises an engineered mutation. In certain embodiments according to (or as applied to) any of the above embodiments, the engineered mutation in the CH3 domain of the antibody heavy chain constant region is T366Y, T366W, T366S, L368A, T394W, T394S, F405A, F405W, Y407T, Y407V or Y407A. In certain embodiments according to (or as applied to) any of the above embodiments, the engineered mutation in the CH3 domain of the antibody heavy chain constant region is Y407V, and wherein the CH3 domain comprises the amino acid sequence shown in SEQ ID NO:6 (DGSFFLVS). In certain embodiments according to (or as applied to) any of the above embodiments, the digestion produces a peptide fragment comprising (such as consisting of) the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8) or GSFFLVSKLTVD (SEQ ID NO: 9). In certain embodiments according to (or as applied to) any of the above embodiments, the sample is digested with trypsin, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7). In certain embodiments according to (or as applied to) any of the above embodiments, the sample is digested with Asp-N, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence DGSFFLVSKLTV (SEQ ID NO: 8). In certain embodiments according to (or as applied to) any of the above embodiments, the sample is digested with Glu-C, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence GSSFFLSKLTVD (SEQ ID NO: 9). In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments according to (or as applied to) any of the above embodiments, the engineered mutation in the CH3 domain of the antibody heavy chain constant region is N434S. In certain embodiments according to (or as applied to) any of the above embodiments, the sample is digested with Glu-C and trypsin, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence ALHSHYTQK (SEQ ID NO: 11).In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0015] In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprising a part of the constant region of the heavy chain of an antibody is an antibody, an Fc fusion protein or an immunoadhesin. In certain embodiments according to (or as applied to) any of the above embodiments, the polypeptide comprising a part of the constant region of the heavy chain of an antibody is an antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a therapeutic antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a monospecific antibody, a bispecific antibody, a trispecific antibody or a multispecific antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a trispecific antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the trispecific antibody comprises four polypeptide chains forming three antigen binding sites, and the three antigen binding sites specifically bind to one or more antigen targets or target proteins, wherein the first polypeptide comprises a structure shown by the following formula: VL2-L1-VL1-L2-CL; the second polypeptide chain comprises a structure shown by the following formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3; the third polypeptide chain comprises a structure shown by the following formula: VH3-CH1-hinge-CH2-CH3; the fourth polypeptide chain comprises a structure shown by the following formula: VL3-CL, wherein VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the first and second polypeptides form a crossover light chain-heavy chain pair; and wherein the second polypeptide chain or the third polypeptide chain comprises the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8) or GSFFLVSKLTVD (SEQ ID NO: 9). In certain embodiments according to (or applied to) any of the above embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:12; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:13; the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:14, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:15.

[0016] In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is conjugated to a drug or a marker. In certain embodiments according to (or as applied to) any of the above embodiments, the drug is selected from a chemotherapeutic agent, a cytotoxic agent or a growth inhibitor. In certain embodiments according to (or as applied to) any of the above embodiments, the marker is a radioisotope, a fluorescent dye or an enzyme. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a human IgG antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the IgG antibody is a human IgG1 antibody or a human IgG4 antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody binds A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 / VTCN1, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 / MCP-1, CCL3 / MIP-1a, CCL4 / MIP-1b, CCL5 / RANTES, CCL7 / MCP-3, C CL8 / mcp-2, CCL11 / eotaxin, CCL15 / MIP-1d, CCL17 / TARC, CCL19 / MIP-3b, CCL20 / MIP-3a, CCL21 / MIP-2, CCL24 / MPIF-2 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCR3, CCR4, CD3, CD19, CD20, CD23 / FCER2, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 / B7-1, CD86 / B7-2, CD122, CD137 / 41BB, CD137L, CD152 / CTLA4, CD154 / CD40L, CD160, CD272, CD273 / PDL2, CD274 / PDL1, CD275 / B7H2, CD276 / B7H3, CD278 / ICOS, CD279 / PD-1, CDH1 / E-cadherin, chitinase, C LEC9, CLEC91, CRTH2, CSF-1 / M-CSF, CSF-2 / GM-CSF, CSF-3 / GCSF, CX3CL1 / SCYD1, CXCL12 / SDF1, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM,ICOLSG, IDO, IFNα, IgE, IGF1R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb / IL25, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 / b4 integrin, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, class II MHC, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 / IL33 receptor, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP / IL7Ra, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM or XCR / GPR5 / CCXCR1. In certain embodiments according to (or as applied to) any of the above embodiments, the method is used to perform pharmacokinetic studies on the polypeptide comprising the constant region of the heavy chain of the antibody in mice, non-human primates and humans. ,

[0017] Unless clearly indicated to the contrary, each embodiment described herein may be combined with one or more other embodiments. In particular, unless clearly indicated to the contrary, any feature or embodiment indicated as preferred or advantageous may be combined with any other one or more features or embodiments indicated as preferred or advantageous. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

[0018] Specifically, the present invention includes but is not limited to the following:

[0019] 1. A method for quantifying the amount of a polypeptide comprising a portion of an antibody heavy chain constant region in a sample, the method comprising:

[0020] (a) digesting the sample comprising a polypeptide comprising a portion of an antibody heavy chain constant region, wherein the portion of the antibody heavy chain constant region comprises an engineered mutation, and wherein the digestion produces peptide fragments derived from the antibody heavy chain constant region, the peptide fragments being 5-26 amino acids in length and comprising the engineered mutation,

[0021] (b) analyzing the digested sample by mass spectrometry to determine the amount of the peptide fragments, thereby determining the amount of the polypeptide comprising a portion of the antibody heavy chain constant region in the sample.

[0022] 2. The method according to item 1, wherein the peptide fragment does not contain methionine (M) or cysteine ​​(C).

[0023] 3. The method according to item 1 or item 2, wherein the peptide fragment does not contain asparagine (N) followed by glycine (G) or serine (S).

[0024] 4. The method according to any one of items 1-3, further comprising purifying and concentrating the digested sample prior to the mass spectrometry analysis.

[0025] 5. A method according to claim 4, wherein the digested sample is purified and concentrated by solid phase extraction (SPE).

[0026] 6. A method according to claim 4, wherein the digested sample is purified and concentrated by SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).

[0027] 7. The method according to any one of items 1-6, wherein the sample is a whole blood sample, a serum sample, a plasma sample or a tissue sample.

[0028] 8. A method according to any one of items 1-7, wherein the sample is from a mouse, a non-human primate or a human.

[0029] 9. A method according to claim 8, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey.

[0030] 10. The method according to any one of items 1-9, wherein the sample is digested with at least one enzyme.

[0031] 11. The method of claim 9, wherein the at least one enzyme is trypsin, chymotrypsin, glutamyl endopeptidase, lysyl endopeptidase, Asp-N, Arg-C, Glu-C, cyanogen bromide (CnBr), or a combination thereof.

[0032] 12. A method according to any one of items 1-11, wherein the mass spectrometry is liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0033] 13. A method according to any one of items 1-12, wherein the polypeptide comprises a CH1 domain, and wherein the CH1 domain comprises the engineered mutation.

[0034] 14. A method according to any one of items 1-12, wherein the polypeptide comprises a CH2 domain, and wherein the CH2 domain comprises the engineered mutation.

[0035] 15. A method according to any one of items 1-12, wherein the polypeptide comprises a CH3 domain, and wherein the CH3 domain comprises the engineered mutation.

[0036] 16. A method according to claim 15, wherein the engineered mutation in the CH3 domain of the antibody heavy chain constant region is T366Y, T366W, T366S, L368A, T394W, T394S, F405A, F405W, Y407T, Y407V or Y407A.

[0037] 17. A method according to claim 16, wherein the engineered mutation in the CH3 domain of the antibody heavy chain constant region is Y407V, and wherein the CH3 domain comprises the amino acid sequence shown in SEQ ID NO:6 (DGSFFLVS).

[0038] 18. A method according to item 17, wherein the digestion produces peptide fragments comprising the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8) or GSFFLVSKLTVD (SEQ ID NO: 9).

[0039] 19. A method according to item 18, wherein the sample is digested with trypsin, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7).

[0040] 20. A method according to item 18, wherein the sample is digested with Asp-N, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence DGSFFLVSKLTV (SEQ ID NO: 8).

[0041] 21. A method according to item 18, wherein the sample is digested with Glu-C, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence GSFFLVSKLTVD (SEQ ID NO: 9).

[0042] 22. The method according to any one of items 16 to 21, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0043] 23. A method according to claim 15, wherein the engineered mutation in the CH3 domain of the antibody heavy chain constant region is N434S.

[0044] 24. A method according to item 23, wherein the sample is digested with Glu-C and trypsin, and wherein the digestion produces a peptide fragment consisting of the amino acid sequence ALHSHYTQK (SEQ ID NO: 11).

[0045] 25. The method according to item 23 or 24, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0046] 26. A method according to any one of items 1 to 25, wherein the polypeptide comprising a portion of an antibody heavy chain constant region is an antibody, an Fc fusion protein or an immunoadhesin.

[0047] 27. A method according to claim 26, wherein the polypeptide comprising a portion of an antibody heavy chain constant region is an antibody.

[0048] 28. A method according to claim 27, wherein the antibody is a therapeutic antibody.

[0049] 29. The method of item 27 or 28, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0050] 30. The method according to any one of items 27-29, wherein the antibody is a monospecific antibody, a bispecific antibody, a trispecific antibody or a multispecific antibody.

[0051] 31. A method according to claim 30, wherein the antibody is a trispecific antibody.

[0052] 32. A method according to claim 31, wherein the trispecific antibody comprises four polypeptide chains forming three antigen binding sites, wherein the three antigen binding sites specifically bind to one or more antigen targets or target proteins, wherein the first polypeptide comprises a structure represented by the following formula: V L2 -L1-V L1 -L2-CL; the second polypeptide chain comprises a structure represented by the following formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 ; The third polypeptide chain comprises a structure represented by the following formula: V H3 -C H1 -Hinge-C H2 -C H3 ; The fourth polypeptide chain comprises a structure represented by the following formula: V L3 -CL, where V L1 is the first immunoglobulin light chain variable domain; V L2V is the second immunoglobulin light chain variable domain; L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 Immunoglobulin C H1 heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; wherein the first and second polypeptides form a crossover light chain-heavy chain pair; and wherein the second polypeptide chain or the third polypeptide chain comprises the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8) or GSFFLVSKLTVD (SEQ ID NO: 9).

[0053] 33. A method according to claim 32, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:12; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:13; the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:14, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:15.

[0054] 34. A method according to any one of items 27-33, wherein the antibody is conjugated to a drug or a marker.

[0055] 35. A method according to claim 34, wherein the drug is selected from a chemotherapeutic agent, a cytotoxic agent or a growth inhibitory agent.

[0056] 36. A method according to claim 35, wherein the marker is a radioactive isotope, a fluorescent dye or an enzyme.

[0057] 37. A method according to any one of items 27-36, wherein the antibody is a human IgG antibody.

[0058] 38. A method according to claim 37, wherein the IgG antibody is a human IgG1 antibody or a human IgG4 antibody.

[0059] 39. A method according to any one of items 27-32 and 34-38, wherein the antibody binds A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 / VTCN1, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 / MCP-1, CCL3 / MIP-1a, CCL4 / MIP-1b, CCL5 / RANTES, CCL7 / MCP-3, CCL8 / mcp-2, CCL11 / eotaxin, CCL15 / MIP-1d, CCL17 / TARC, CCL 19 / MIP-3b, CCL20 / MIP-3a, CCL21 / MIP-2, CCL24 / MPIF-2 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCR3, CCR4, CD3, CD19, CD20, CD23 / FCER2, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 / B7-1, CD86 / B7-2, CD122, CD137 / 41BB, CD137L, CD152 / CTLA4, CD154 / CD40L, CD160, CD272, CD273 / PDL 2. CD274 / PDL1, CD275 / B7H2, CD276 / B7H3, CD278 / ICOS, CD279 / PD-1, CDH1 / E-cadherin, chitinase, CLEC9, CLEC91, CRTH2, CSF-1 / M-CSF, CSF-2 / GM-CSF, CSF-3 / GCSF, CX3CL1 / SCYD1, CXCL12 / SDF1, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2 , HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10 , rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb / IL25, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 / b4 integrin, ITK, KIR, LAG3, LAMP1, leptin,LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 / IL33 receptor, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP / IL7Ra, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, or XCR / GPR5 / CCXCR1.

[0060] 40. A method according to any one of items 1-39, wherein the method is used for pharmacokinetic studies of the polypeptide comprising an antibody heavy chain constant region in mice, non-human primates and humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Sequence alignments of the Fc regions of several exemplary therapeutic antibodies are shown. Predicted tryptic peptides containing at least one amino acid substitution are underlined. Cysteines present within tryptic peptides are indicated by bold arrows. SEQ ID NOs: 2 and 5 include knob mutation T366W. SEQ ID NOs: 3 and 4 include hole mutations T366S, L368A, and Y407V.

[0062] Figures 2A-2C Shown are the results of LC-MS / MS analysis of a tryptic digest of a trispecific construct (TRI-1) comprising heavy chains having the amino acid sequences set forth in SEQ ID NOs: 2 and 3. SEQ ID NO: 2 comprises the engineered "knob" mutation T366W and SEQ ID NO: 3 comprises the engineered "hole" mutations T366S, L368A and Y407V. Tryptic digestion of SEQ ID NO: 3 is predicted to produce a peptide having the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), i.e., the "engineered TTPP peptide". Figure 2A The extracted ion chromatogram of m / z 905.40-907.60 is shown. Figure 2B Total ion chromatograms are shown. Figure 2C MS / MS spectra of engineered TTPP peptides are shown.

[0063] Figures 3A-3D Trypsin-digested mouse serum ( Figure 3A ), monkey serum ( Figure 3B ), human serum ( Figure 3C) and TRI-1 in PBST ( Figure 3D ). The run time of the engineered TTPP is indicated by the boxes.

[0064] Figures 4A-4D Mouse serum ( Figure 4A ), monkey serum ( Figure 4B ), human serum ( Figure 4C ) and PBST( Figure 4D ) Extracted ion chromatogram of TRI-1 digested with trypsin in ). The run time of the engineered TTPP is indicated by the box.

[0065] Figures 5A-5C The extracted ion chromatogram of engineered TTPP from trypsin-digested TRI-1 in PBST is shown, m / z = 905.36-905.55. TRI-1 concentration was 20 μg / mL ( Figure 5A )、2μg / mL( Figure 5B ) and 0.2 μg / mL ( Figure 5C The peak areas of TTPP peptide are 44064886 (Figure A), 2633166 ( Figure 5B ) and 368649( Figure 5C ).

[0066] Figures 6A-6C The extracted ion chromatogram of unlabeled FNWYVDGVEVHNAK (SEQ ID NO: 10) (FNWY) from trypsin-digested TRI-1 in PBST is shown, m / z = 559.90-559.98. The TRI-1 concentration was 20 μg / mL ( Fig. 6A )、2μg / mL( Figure 6B ) and 0.2 μg / mL ( Figure 6C The peak area of ​​FNWY peptide is 70091279 ( Fig. 6A )、1078300( Figure 6B ) and 209110( Figure 6C ).

[0067] Figures 7A-7C The extracted ion chromatogram of isotopically labeled FNWY (FNWY(heavy)) from trypsin-digested SILUMAB in PBST, which was used as an internal standard for TRI-1 digestion, m / z=562.58-562.63. TRI-1 concentration was 20 μg / mL ( Fig. 7A )、2μg / mL( Figure 7B ) and 0.2 μg / mL ( Figure 7C The peak area of ​​FNWY peptide is 1369078 ( Fig. 7A )、343482( Figure 7B ) and 473743( Figure 7C ).

[0068] Figures 8A-8D The extracted ion chromatogram of engineered TTPP from trypsin-digested TRI-1 in mouse serum is shown, m / z=905.39-905.55. TRI-1 concentration was 0 μg / mL (control) ( Fig. 8A )、0.2μg / mL( Figure 8B )2μg / mL( Figure 8C ) and 20 μg / mL ( Fig.8D ). The peak area of ​​TTPP peptide is ND (not detected) ( Fig. 8A )、ND(Not Detected)( Figure 8B )、2168471( Figure 8C ) and 11833127( Fig.8D ).

[0069] Figures 9A-9D The extracted ion chromatogram of unlabeled FNWY from trypsin-digested TRI-1 in mouse serum is shown, m / z = 559.90-559.98. The TRI-1 concentration was 0 μg / mL (control) ( Fig.9A )、0.2μg / mL( Fig. 9B )、2μg / mL( Fig. 9C ) and 20 μg / mL ( Fig.9D The peak area of ​​FNWY peptide is ND (not detected) ( Fig.9A )、410330( Fig. 9B )、2500109( Fig. 9C ) and 22039621( Fig.9D ).

[0070] Figures 10A-10D Shown is the extracted ion chromatogram of FNWY (heavy) from trypsin-digested SILUMAB in mouse serum, which was used as an internal standard for TRI-1 digestion, m / z = 562.58-562.63. TRI-1 concentration was 0 μg / mL (control) ( Fig. 10A )、0.2μg / mL( Fig. 10B )、2μg / mL( Fig. 10C ) and 20 μg / mL ( Fig. 10D The peak area of ​​FNWY peptide is 1184181 ( Fig. 10A )、2310675( Fig. 10B )、1199642( Fig. 10C ) and 967309( Fig. 10D ).

[0071] Figures 11A-11D The extracted ion chromatogram of the engineered TTPP from trypsin-digested TRI-1 in monkey serum is shown, m / z=905.39-905.55. The TRI-1 concentration was 0 μg / mL (control) ( Fig.11A )、0.2μg / mL( Fig. 11B )、2μg / mL( Fig. 11C ) and 20 μg / mL ( Fig.11D ). The peak area of ​​TTPP peptide is ND (not detected) ( Fig.11A )、ND(Not Detected)( Fig. 11B )、1013964( Fig. 11C ) and 3751017( Fig.11D ).

[0072] Figures 12A-12D The extracted ion chromatogram of unlabeled FNWY from trypsin-digested TRI-1 in monkey serum is shown, m / z = 559.92-559.95. The TRI-1 concentration was 0 μg / mL (control) ( Fig. 12A )、0.2μg / mL( Fig. 12B )、2μg / mL( Fig. 12C ) and 20 μg / mL ( Fig.12D The peak area of ​​FNWY peptide is 75675 ( Fig. 12A )、97731( Fig. 12B )、1298150( Fig. 12C ) and 13378187( Fig.12D ).

[0073] Figures 13A-13D Shown is the extracted ion chromatogram of FNWY (heavy) from trypsin-digested SILUMAB in monkey serum, which was used as an internal standard for TRI-1 digestion, m / z = 562.58-562.63. TRI-1 concentration was 0 μg / mL (control) ( Fig.13A )、0.2μg / mL( Fig. 13B )、2μg / mL( Fig. 13C ) and 20 μg / mL ( Fig.13D The peak area of ​​FNWY peptide is 1446203 ( Fig.13A )、1216254( Fig. 13B )、1271728( Fig. 13C ) and 1349983( Fig.13D ).

[0074] Figures 14A-14DThe extracted ion chromatogram of engineered TTPP from trypsin-digested TRI-1 in human serum is shown, m / z=905.39-905.55. TRI-1 concentration was 0 μg / mL (control) ( Fig.14A )、0.2μg / mL( Fig. 14B )、2μg / mL( Fig. 14C ) and 20 μg / mL ( Fig.14D ). The peak area of ​​TTPP peptide is ND (not detected) ( Fig.14A )、ND(Not Detected)( Fig. 14B )、194065( Fig. 14C ) and 1848332( Fig.14D ).

[0075] Figures 15A-15D Comparison of area ratios versus antibody concentrations is shown. Fig.15A Shown is a comparison of the area ratio of engineered TTPP:FNWY(Heavy) in PBST relative to the antibody concentration of TRI-1. Fig. 15B Shown is a comparison of the area ratio of unlabeled FNWY:FNWY(Heavy) in PBST relative to the antibody concentration of TRI-1. Fig. 15C Shown is a comparison of the area ratio of engineered TTPP:FNWY(Heavy) in mouse serum relative to TRI-1 antibody concentration. Fig.15D A comparison of the area ratio of engineered TTPP:FNWY(heavy) relative to antibody concentration of TRI-1 in monkey serum is shown.

[0076] Fig.16 Shown are the sequences of the heavy and light chains of exemplary trispecific antibodies quantified in Example 2. The sequences of the surrogate peptides quantified in the LC-MS / MS assay are underlined.

[0077] Fig.17 A schematic diagram of an exemplary trispecific antibody is shown, and wherein Fig.16 The sequences of the alternative peptides underlined in are located in the trispecific antibodies.

[0078] Fig.18 An exemplary workflow diagram of a method for quantifying trispecific antibodies using TTPP peptides is provided.

[0079] Fig.19A An example chromatogram of the TTPP peptide at LLOQ (lower limit of quantitation, 2.5 μg / ml) is shown. Fig.19B Stable isotope labeled TTPP peptide (standard [ 13 C11- 15N2] -LTTPPVLDSDGSFFLVSK (SEQ ID NO: 20)), the TTPP peptide used as an internal standard in Example 3. Fig.19C A calibration curve for TTPP peptide in the range of 2.5 μg / mL - 10,000 μg / mL is shown.

[0080] Fig. 20A Serum calibration curves analyzed using linear regression analysis with 1 / x weighting are shown. Fig. 20B A second serum calibration curve using linear regression analysis with 1 / x weighting is shown. Fig. 20A and 20B A sample of the curve in .

[0081] Fig.21A Shown is a comparison of the relative levels of the trispecific antibodies as detected by measuring the levels of TTPP peptide in sera obtained from mice administered the trispecific antibodies by LC-MS / MS. Fig.21B Shown is a comparison of the relative levels of trispecific antibodies as detected via ELISA in sera obtained from mice administered with the trispecific antibodies.

[0082] Fig.22A Shown are the results of LC-MS / MS analysis of blank rat serum samples. Fig. 22B Shown is the MS / MS spectrum of a rat serum sample spiked with 5 μg / ml of the trispecific antibody. Fig. 22C Shown are the results of LC-MS / MS analysis of blank monkey serum samples. Fig.22D Shown is the MS / MS spectrum of a monkey serum sample spiked with 5 μg / ml of the trispecific antibody. Fig.22E Shown are the results of LC-MS / MS analysis of blank monkey serum samples. Fig.22F Shown is the MS / MS spectrum of a human serum sample spiked with 5 μg / ml of the trispecific antibody. DETAILED DESCRIPTION

[0083] definition

[0084] As used herein, "antibody constant region" refers to the more conserved regions of an antibody, e.g., outside the variable domains. The term may include the light chain constant region, i.e., the CL domain, the hinge region, and the heavy chain constant domains CH1, CH2, CH3, and optionally CH4.

[0085] As used herein, the term "engineered mutation" refers to a mutation that is designed by a human (ie, the mutation does not occur spontaneously due to natural causes and / or is the result of deliberate human manipulation).

[0086] As used herein, the term "antibody" may refer to intact antibodies, antibody fragments comprising at least a portion of the heavy chain constant region (including but not limited to Fab, F(ab')2, Fab'-SH, Fv, scFv or single heavy chain antibodies), provided that they exhibit the desired biological activity; monoclonal antibodies; polyclonal antibodies; monospecific antibodies; multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies); and antibody-like proteins.

[0087] The term "antibody" generally refers to a heterotetrameric complex comprising two light (L) chains and two heavy (H) chains. In addition to intrachain disulfide bridges, a variable number of disulfide bonds connect the two heavy chains, and one disulfide bond connects each light chain to the heavy chain. The heavy chain includes a variable domain (VH), followed (N-terminal to C-terminal) by three or four constant domains. The light chain includes a variable domain (VL), followed by a constant domain (CL). Typically, mammalian light chains belong to one of two classes based on the amino acid sequence: κ and λ.

[0088] As used herein, the term "multispecific" when used to refer to an antibody or antibody fragment includes antibodies or antibody fragments with two or more different binding specificities (e.g., bispecific and trispecific antibodies). For example, each binding specificity may recognize a different antigen, or each binding specificity may recognize the same antigen with different affinities and / or precise epitopes. In some embodiments, each different binding specificity comprises one or more different antibody antigen binding domains (e.g., variable domains), such that the multispecific antibody or antibody fragment comprises, for example, a first antigen binding domain with a first binding specificity, a second antigen binding domain with a binding domain with a second binding specificity, and so on. Various exemplary multispecific antibody formats (e.g., bispecific and trispecific antibody formats) are known in the art and are described in further detail elsewhere herein.

[0089] Before describing the disclosed embodiments in detail, it is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0090] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more of said molecules, and so forth.

[0091] As used herein, the term "about" refers to the usual error range for the respective value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments for that value or parameter per se.

[0092] It should be understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0093] Methods for quantifying peptides in samples

[0094] Mass spectrometry (MS) is a feasible alternative to ligand binding assay (LBA) for quantitative analysis of therapeutic proteins in samples obtained from, for example, preclinical animal studies. The typical procedure for MS-based quantification requires digestion of each sample to generate peptide fragments and quantification of the amount of specific peptides derived from the therapeutic protein as a substitute for the therapeutic protein itself. Currently, "universal" surrogate peptides derived from the constant region of human antibodies are used in the universal method of quantitatively quantifying human antibodies, humanized antibodies or chimeric antibodies in non-human animal models. Such universal methods are based on the following principles: the sequence of "universal" surrogate peptides is conserved in all human Fc domains, but does not exist in species (e.g., mice, rats, dogs, monkeys, etc.) for preclinical efficacy and safety studies. However, it is impossible to use "universal" surrogate peptides in samples of human patients to accurately quantify therapeutic proteins such as those comprising the constant region of human antibodies, because the sequence of the "universal" surrogate peptides is also present in endogenous human antibodies.

[0095] Many therapeutic proteins (e.g., antibodies) comprising human antibody constant regions also comprise engineered mutations that have been introduced into the constant region to, for example, modulate effector function, modulate serum half-life, promote heterodimerization, etc. Applicants have found that peptides derived from portions of the constant region containing engineered mutations are unique in both non-human and human proteome contexts. Therefore, the methods described herein can be used to quantify therapeutic proteins in preclinical samples (i.e., obtained from animal species) and clinical samples (i.e., obtained from human patients).

[0096] A method for quantifying the amount of a polypeptide in a sample is provided, wherein the polypeptide comprises a portion of an antibody heavy chain constant region, and wherein a portion of the antibody heavy chain constant region comprises an engineered mutation. In some embodiments, the method comprises the following steps: (a) digesting a sample suspected of comprising a polypeptide to produce a digested sample comprising a peptide fragment containing an engineered mutation; and (b) analyzing the sample digested via mass spectrometry to determine the amount of the peptide fragment comprising the engineered mutation, thereby determining the amount of the polypeptide. Polypeptides (e.g., fusion proteins, immunoadhesins, and antibodies) that can be quantified using the methods described herein are further described in detail elsewhere herein.

[0097] In some embodiments, the methods provided herein are used for pharmacokinetic studies (such as in preclinical studies or clinical trials). In some embodiments, the method comprises the following steps: For example, during preclinical studies, before quantification, a polypeptide comprising a portion of an antibody heavy chain constant region with an engineered mutation is administered to an animal. Exemplary non-human animals to which a polypeptide comprising a portion of an antibody heavy chain constant region with an engineered mutation can be administered are described in detail below. In some embodiments, the method includes administering a polypeptide comprising a portion of an antibody heavy chain constant region with an engineered mutation to a human (e.g., a human patient) during a clinical trial, before quantification.

[0098] In some embodiments, the sample comprising a polypeptide containing a part of the constant region of the heavy chain of an antibody with an engineered mutation is a biological sample derived from an animal, obtained or separated. In some embodiments, the animal derived, obtained or separated sample is an animal to which a polypeptide comprising a part of the constant region of the heavy chain of an antibody with an engineered mutation is applied. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a human (e.g., human patient), a non-human primate (NHP) (e.g., cynomolgus monkey, rhesus monkey, marmoset, silk monkey, spider monkey, owl monkey, squirrel monkey, long-tailed monkey (vervet money), baboon or other) or a rodent (e.g., mouse or rat).

[0099] In some embodiments, the sample analyzed according to the method provided herein is any sample suspected of containing a polypeptide comprising a part of the constant region of the heavy chain of an antibody with an engineered mutation. In some embodiments, the sample is a body fluid or derived from a body fluid, including but not limited to, for example, blood, plasma, serum, milk, bronchial lavage fluid, amniotic fluid, saliva, bile or tears. In some embodiments, the sample comprises tissue or cells. In some embodiments, the sample comprises serum and a known amount of peptides (i.e., "spiked" serum). In some embodiments, the spiked serum also comprises a buffer. In some embodiments, the spiked serum is used as a reference for the amount of a polypeptide quantitatively present in a sample derived from, obtained from or separated from an animal comprising a part of the constant region of the heavy chain of an antibody with an engineered mutation.

[0100] In some embodiments, the method further includes treating the sample prior to digestion, for example, to enrich the sample for a polypeptide comprising a portion of an antibody heavy chain constant region having an engineered mutation, or, for example, to deplete an abundant protein (such as albumin from a blood, serum, or plasma sample) from the sample. In some embodiments, treating the sample includes performing an antibody pull-down assay. Alternatively or additionally, in some embodiments, treating the sample includes performing gel electrophoresis, extraction, precipitation, centrifugation, chromatography (e.g., affinity capture chromatography, size exclusion chromatography, etc.), ultrafiltration, and / or one or more additional separation steps known to those of ordinary skill in the art.

[0101] In some embodiments, the sample is digested by chemical cleavage. Exemplary chemical reagents for cleavage at specific sites of the polypeptide include, but are not limited to, for example, formic acid, hydroxylamine, iodobenzoic acid, and NTCB (2-nitro-5-thiocyanobenzoic acid). In some embodiments, the sample is digested with an enzyme such as an endopeptidase. In some embodiments, the enzyme is a site-specific endopeptidase. Exemplary site-specific proteases include, but are not limited to, trypsin, chymotrypsin (high specificity, cleaving the c-terminus of FYW, not before P), chymotrypsin (low specificity, cleaving the c-terminus of FYWML, not before P), glutamyl endopeptidase, lysyl endopeptidase, Asp-N protease, Arg-C protease, Lys-C protease, Lys-N protease, Staphylococcus aureus V8 (also known as glutamyl endopeptidase or Glu-C protease), cyanogen bromide (CnBr), elastase, pepsin ( In some embodiments, the sample is digested with a non-specific endopeptidase such as papain or proteinase K. In some embodiments, the sample is digested with a mixture comprising two (or more) endopeptidases (including site-specific and / or non-site-specific). In some embodiments, the two or more endopeptidases are used to digest the sample simultaneously. In some embodiments, the two or more endopeptidases are used to digest the sample sequentially. Methods for digesting polypeptides in preparation for analysis by mass spectrometry are well known in the art. Exemplary methods are provided in, for example, Gundry et al. (2009) Curr Protoc Mol Biol. doi: 10.1002 / 0471142727.mb1025s88; Hedrick et al. (2015) Curr Protoc Chem Biol. 7(3): 201-222; Giansanti et al. (2016) Nature Protocols. 11: 993-1006; Nordhoff et al. (Int J Mass Spect. 226(1): 163-180; and Zhang et al. (2014) Curr Protoc Mol Biol. doi: 10.1002 / 0471142727.mb1021s108.

[0102] The digestion of sample produces the peptide fragment comprising engineered mutation.In some embodiments, the length of the peptide fragment produced by digestion is 5 to 40 amino acid, for example, and the length is 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 or 40 amino acid.In some embodiments, the peptide fragment does not include cysteine ​​(Cys) and / or methionine (M) residues.Alternately or additionally, in some embodiments, the peptide does not include the asparagine residue (N) (that is, NG or NS) that is thereafter a glycine residue (G) or a serine residue (S). In some embodiments, the peptide fragments do not contain adjacent endopeptidase cleavage sites, e.g., two or more site-specific endopeptidase cleavage sites separated by 6, 5, 4, 3, 2, or 1 amino acids. A variety of computer software tools have been developed to predict peptide populations that can be generated by digestion (e.g., digestion with an enzyme or chemical cleavage reagent or with a combination of enzymes and / or chemical cleavage reagents). Such tools, including, but not limited to, PChopper, PeptideCutter, MAPPP, IPEP, MS-Digest, and Protein Digestion Simulator, are well known in the art and are publicly available, e.g., on the World Wide Web.

[0103] In some embodiments, the method further includes purifying and / or concentrating the digested sample before mass spectrometry analysis. In some embodiments, purifying and / or concentrating the digested sample includes performing affinity capture chromatography or solid phase extraction (SPE), and eluting the purified and concentrated sample. (See, for example, Gudry et al. (2009) "Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow." Curr Protoc Mol Biol. CHAPTER: Unit 10.25. doi: 10.1002 / 0471142727.mb1025s88). In some embodiments, purifying and / or concentrating the digested sample includes performing stable isotope standards and capturing (SISCAPA) by anti-peptide antibodies. In some embodiments, antibody pull-down assay is performed on the sample, digested (e.g., using one or more endopeptidases known in the art), and purified and / or concentrated by SISCAPA. Details about SISCAPA are provided in, for example, Anderson et al. (2004) "Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J Proteome Res. 3(2): 235-44; U.S. Pat. Nos. 7,632,686 and 9,164,089; Whiteaker et al. (2011) "Evaluation of large scale quantitative proteomicassay development using peptide affinity-based mass spectrometry." Mol Cell Proteomics. 10(4): M110.005645; and Rasavi et al. (2016) "Multiplexed longitudinal measurement of protein biomarkers in DBS using an automated SISCAPA workflow." Bioanalysis. 8(15): 1597-1609.

[0104] In some embodiments, the digested samples are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS is a method in which a sample mixture is first separated by liquid chromatography and then ionized (e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or atmospheric pressure photoionization (APPI)) and characterized by mass-to-charge ratio and relative abundance using two mass spectrometers in series. Details on LC-MS / MS are provided in: e.g. Grebe et al. (2011) "LC-MS / MS in the Clinical Laboratory—Where to From Here?" Clin. Biochem Review. 32(1):5-31; El-Khoury et al. "Liquid Chromatography-Tandem Mass Spectrometry in the Clinical Laboratory." J. Chrom.&Separation Tech.4:e115.doi:10.4172 / 2157-7064.1000e115; Shushan et al. (2010) "A review of clinical diagnostic applications of liquid chromatography–tandem mass spectrometry." Mass Spec.Rev.29:930-944, 2010.

[0105] In some embodiments, the methods provided herein can be used to detect a peptide in a sample, wherein the concentration of the peptide in the sample is about any of 200, 150, 100, 50, 15, 10, 5, 1, 0.5, 0.25, 0.1, 0.075, 0.05, 0.025, or 0.01 ng / ml, including any ranges between these values.

[0106] Engineered mutations in the constant region of the antibody heavy chain

[0107] In some embodiments, the polypeptide quantified according to the method provided herein comprises a CH1 domain (or a portion thereof, e.g., 10-50 amino acids) containing an engineered mutation. In some embodiments, the CH1 domain extends from about residue 114 of the antibody heavy chain to about residue 223, according to the Kabat numbering system. In some embodiments, the CH1 domain extends from about residue 118 of the antibody heavy chain to about residue 215, according to the EU numbering system. See, e.g., the International Immunogenetics Information System (IMGT) Web Resources at WorldWideWeb.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. These amino acid residue positions are based on human IgG; however, it is not intended that the method provided herein be limited to use with a polypeptide comprising a CH1 domain (or a portion thereof) of human IgG. The corresponding CH1 domain sequences from other human Igs, and the corresponding CH1 domain sequences of other mammals (e.g., macaques, cynomolgus monkeys, mice, rats, etc.) are publicly available.

[0108] In certain embodiments, the engineered mutations in the CH1 domain (or a portion thereof) comprise (e.g., consist of) amino acid substitutions, insertions, or deletions that do not affect (or substantially affect) the desired activity of a polypeptide comprising an antibody heavy chain constant region. In some embodiments, the substitution or insertion comprises a substitution or insertion of an unnatural amino acid or a conjugated amino acid. Amino acid substitutions, insertions, or deletions can be introduced into the CH1 domain (or a portion thereof) by altering the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis) or by peptide synthesis, as described in further detail elsewhere herein.

[0109] In certain embodiments, the polypeptide quantified according to the methods provided herein comprises a human IgG1 CH1 domain (or a portion thereof). In some embodiments, the engineered mutation in the human IgG1 CH1 domain (or a portion thereof) drives Fc heterodimerization (e.g., for producing bispecific antibodies, multispecific antibodies, or one-armed antibodies). In some embodiments, the engineered mutation in the human IgG1 CH1 domain (or a portion thereof) is an amino acid substitution at K147 or K213 (according to the EU numbering system). In some embodiments, the engineered mutation in the human IgG1 CH1 domain comprises an amino acid substitution (e.g., consisting of an amino acid substitution): K147D, K147E, K213D, or K213E (according to the EU numbering system).

[0110] In certain embodiments, the polypeptide quantified according to the method provided herein comprises a CH2 domain (or a portion thereof, e.g., 10-50 amino acids) containing an engineered mutation. In some embodiments, the CH2 domain of human IgG extends from about residue 231 of the antibody heavy chain to about residue 340, according to the EU numbering system. See, for example, the International Immunogenetics Information System (IMGT) web resource at WorldWideWeb.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. These amino acid positions are based on human IgG; however, this is not intended to limit the method provided herein to use with a polypeptide comprising a CH2 domain (or a portion thereof) of human IgG. The corresponding CH2 domain sequences from other human Igs are publicly available, and the corresponding CH2 domain sequences of other mammals (e.g., macaques, cynomolgus monkeys, rats, mice, etc.) are also publicly available. In certain embodiments, the engineered mutations in the CH2 domain (or a portion thereof) include amino acid substitutions, insertions, or deletions that do not affect (or substantially do not affect) the desired activity of a polypeptide comprising an antibody heavy chain constant region. In some embodiments, the substitution or insertion comprises substitution or insertion of an unnatural amino acid or a conjugated amino acid.

[0111] In certain embodiments, the polypeptide quantified according to the methods provided herein comprises a human IgG1 CH2 domain (or a portion thereof). In some embodiments, the engineered mutations in the human IgG1 CH2 domain (or a portion thereof) modulate effector function. In some embodiments, the engineered mutations in the human IgG1 CH2 domain (or a portion thereof) comprise amino acid substitutions at one (or more) of the following residues: E233, L234, L235, G236, P238, S239, F243, T250, M252, S254, T256, P257, S267, R292, Q295, N297, S298, T299, Y300, Q311, K322, A327, L328, P329, A330, P331, I332, and E333 (according to the EU numbering system). In some embodiments, human IgG1 The engineered mutation in the CH2 domain (or portion thereof) comprises one (or more) of the following amino acid substitutions: E233P, L234V, L234A, L235V, L235A, G236A, P238D, S239D, F243L, T250Q, T250R, M252Y, S254T, T256E, P257I, S267E, R292P, Q295R, N297Q, N297D, N297 332Y, E333A and E333S (according to the EU numbering system). Alternatively or additionally, the engineered mutation in the human IgG1 CH2 domain (or portion thereof) comprises (e.g., consists of) ΔG236 (according to the EU numbering system).

[0112] In certain embodiments, the polypeptide quantified according to the methods provided herein comprises a human IgG2 CH2 domain (or a portion thereof). In some embodiments, the engineered mutation in the human IgG2 CH2 domain (or a portion thereof) modulates effector function. In some embodiments, the engineered mutation in the human IgG2 CH2 domain (or a portion thereof) comprises an amino acid substitution at one or more of the following residues: K326 and E333 (according to the EU numbering system). In some embodiments, the engineered mutation in the human IgG1 CH2 domain (or a portion thereof) comprises one (or more) of the following amino acid substitutions: K326W and E333S (according to the EU numbering system).

[0113] In certain embodiments, the polypeptide quantified according to the methods provided herein comprises a human IgG3 CH2 domain. In some embodiments, the engineered mutations in the human IgG3 CH2 domain regulate effector function. In some embodiments, the engineered mutations in the human IgG3 CH2 domain comprise an amino acid substitution at E235 (according to the EU numbering system). In some embodiments, the amino acid substitution comprises E235Y.

[0114] In certain embodiments, the polypeptide to be quantified according to the methods provided herein comprises a human IgG4 CH2 domain (or a portion thereof). In some embodiments, the engineered mutations in the human IgG4 CH2 domain (or a portion thereof) regulate effector functions. In some embodiments, the engineered mutations in the human IgG4 CH2 domain (or a portion thereof) comprise amino acid substitutions at one (or more) of the following residues: S228, F234, L235, F296, G327, and P329 (according to the EU numbering system). In some embodiments, the amino acid substitutions include one (or more) of the following amino acid substitutions: S228P, F234A, F234L, L235A, F296Y, G327A, P329G, and P329N.

[0115] In some embodiments, the polypeptide to be quantified according to the methods provided herein comprises a CH3 domain (or a portion thereof, e.g., 10-50 amino acids) containing an engineered mutation. In some embodiments, according to the EU numbering system, the CH3 domain extends from about residue 341 to about residue 447. See, e.g., International Immunogenetics Information System (IMGT) Web Resources on WorldWideWeb.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. These amino acid positions are based on human IgG; however, it is not intended that the methods provided herein be limited to use with polypeptides comprising the CH3 domains of human IgG. The corresponding CH3 domain sequences from other human Igs are publicly available, and the corresponding CH3 domain sequences from other Igs of other mammals (e.g., macaques, cynomolgus monkeys, rats, mice, etc.) are also publicly available. In certain embodiments, the engineered mutations in the CH3 domain (or a portion thereof) are amino acid substitutions, insertions, or deletions that do not affect (or substantially do not affect) the desired activity of the polypeptide comprising the heavy chain constant region of the antibody. In some embodiments, the substitution or insertion comprises substitution or insertion of an unnatural amino acid or a conjugated amino acid.

[0116] In certain embodiments, the polypeptide to be quantified according to the methods provided herein comprises a human IgG4 CH3 domain (or a portion thereof). In some embodiments, the engineered mutation in the human IgG1 CH4 domain (or a portion thereof) modulates effector function. In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) comprises (e.g., consisting of) an amino acid substitution at residue R409 (according to the EU numbering system). In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) comprises (e.g., consisting of) an amino acid substitution R409K.

[0117] In certain embodiments, the polypeptide to be quantified according to the method provided herein comprises a human IgG1 CH3 domain (or a portion thereof). In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) regulates effector function and / or improves serum half-life. In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) comprises one or more amino acid substitutions of the following residues: N343, E380, E382, P396, M428, H433, N434, and Y436 (according to the EU numbering system). In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) comprises one (or more) of the following amino acid substitutions: N343A, E380A, E382V, P396L, M428I, M428L, H433K, N434S, N434F, and Y436H (according to the EU numbering system). In some embodiments, the engineered mutations in the human IgG1 CH3 domain (or portion thereof) drive Fc heterodimerization (e.g., for the production of bispecific antibodies, multispecific antibodies, or one-armed antibodies). In some embodiments, the engineered mutations in the human IgG1 CH3 domain (or portion thereof) comprise amino acid substitutions at one (or more) of the following residues: Y349, S354, R355, D356, E357, K360, T366, L368, K370, K392, T394, D399, F405, Y407, K409, and K439 (according to the EU numbering system). In some embodiments, the human IgG1 The engineered mutations in the CH3 domain (or portion thereof) comprise one (or more) of the following amino acid substitutions: Y349C, S354C, R355D, R355E, D356K, D356R, E357K, E357R, K360D, K360E, T366R, T366K, T366N, T366Q, T366Y, T366W, T366S, T366E, T366G, L368A, L368K, L368Q, L368D, L368E, L368G, L368H, L368I, L368N, L368R, L368S, L368T , L368V, L368W, K370W, K370D, K370E, K392D, K392E, T394W, T394S, D399A, D399G, D399I, D399L, D399M, D399N, D299S, D399T, D399F, D399H, D399K, D399R, D399Y, F405A, F405W, Y407T, Y407V, Y407A, K409R, K409A, K409H, K409D, K409E, K409G, K439D and K439E (according to the EU numbering system).

[0118] In some embodiments, the engineered mutation in the human IgG1 CH3 domain (or a portion thereof) comprises Y407V (e.g., consisting of Y407V). In some embodiments, the polypeptide comprising the heavy chain constant region of the antibody having the engineered mutation comprises the amino acid sequence (DGSFFLVS) shown in SEQID NO:6. In some embodiments, digestion of a sample comprising (or suspected of comprising) a polypeptide comprising the heavy chain constant region of the antibody having the engineered Y407V mutation produces a peptide fragment comprising (e.g., consisting of) the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO:7), DGSFFLVSKLTV (SEQ ID NO:8), or GSFFLVSKLTVD (SEQ ID NO:9). In some embodiments, a sample comprising (or suspected of comprising) a polypeptide comprising the heavy chain constant region of the antibody having the engineered Y407V mutation is digested with trypsin, and digestion produces a peptide fragment comprising (e.g., consisting of) the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO:7). In some embodiments, a sample comprising (or suspected of comprising) a polypeptide comprising an antibody heavy chain constant region having an engineered Y407V mutation is digested with Asp-N, and the digestion produces a peptide fragment comprising (e.g., consisting of) the amino acid sequence DGSFFLVSKLTV (SEQ ID NO: 8). In some embodiments, a sample comprising (or suspected of comprising) a polypeptide comprising an antibody heavy chain constant region having an engineered Y407V mutation is digested with Glu-C, and the digestion produces a peptide fragment comprising (e.g., consisting of) the amino acid sequence GSFFLVSKLTVD (SEQ ID NO: 9).

[0119] In some embodiments, the engineered mutation in the human IgG1 CH3 domain comprises N434S (e.g., consisting of N434S). In some embodiments, a sample comprising (or suspected of comprising) a polypeptide comprising an antibody heavy chain constant region having an engineered N434S mutation is digested with Glu-C and trypsin, and the digestion produces a peptide fragment comprising (e.g., consisting of): a peptide fragment of the amino acid sequence ALHSHYTQK (SEQ ID NO: 11).

[0120] In certain embodiments, engineered mutations are introduced into the antibody heavy chain constant region (or portion thereof) by standard molecular biology techniques known in the art. A variety of genetic engineering methods have been previously described. Such mutagenesis methods include, but are not limited to, for example, error-prone PCR, loop shuffling, oligonucleotide-directed mutagenesis, random nucleotide insertion, or other methods before recombination. Further details about these methods are described in, for example, Abou-Nadler et al. (2010) Bioengineered Bugs.1, 337-340; Firth et al. (2005) Bioinformatics.21, 3314-3315; Cirino et al. (2003) Methods Mol Biol.231, 3-9; Pirakitikulr (2010) Protein Sci.19, 2336-2346; Steffens et al. (2007) J.BiomolTech.18, 147-149; and others.

[0121] A polypeptide comprising a portion of an antibody heavy chain constant region

[0122] Any polypeptide comprising a part of the constant region of the heavy chain of an antibody can be used to carry out the method provided herein, wherein a part of the constant region of the heavy chain of the antibody comprises an engineered mutation. In some embodiments, the polypeptide comprises all or part of a CH1 domain, a CH2 domain and / or a CH3 domain (e.g., 10 to 50 amino acids), provided that the domain (or part thereof) comprises an engineered mutation (e.g., an engineered mutation described in other places herein). In some embodiments, a part of the heavy chain constant domain comprising an engineered mutation comes from or is derived from a mammal (e.g., a human, a non-human primate, a mouse, a rat, etc.). In some embodiments, a part of the heavy chain constant domain comprising an engineered mutation comes from or is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4), human IgA (e.g., IgA1 or IgA2), human IgM, human IgE, or human IgD. In some embodiments, the portion of the heavy chain constant region comprising the engineered mutation is from a mouse or derived from a mouse (such as a mouse IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgM antibody heavy chain constant region).

[0123] In certain embodiments, the polypeptide comprising a portion of an antibody heavy chain constant region having an engineered mutation is an antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a constant region, such as a constant region of human IgG1, human IgG2, human IgG3, human IgG4, human IgA1, human IgA2, human IgM, human IgE, or human IgD, comprising at least one engineered mutation.

[0124] In certain embodiments, the polypeptide comprising a portion of an antibody heavy chain constant region having an engineered mutation is a fusion polypeptide. In some embodiments, the fusion polypeptide is an Fc fusion polypeptide comprising an Fc domain (i.e., CH2 and CH3 domain) of an antibody heavy chain constant region. In some embodiments, the fusion polypeptide is an immunoadhesin, i.e., a fusion polypeptide in which a functional domain of a binding protein (e.g., a receptor, a ligand, or a cell adhesion polypeptide) is fused to a portion of an antibody heavy chain constant region (usually a hinge and an Fc domain).

[0125] Antibody

[0126] In some embodiments, the polypeptide comprising a part of the constant region of the heavy chain of an engineered mutant antibody is an antibody. In some embodiments, the antibody is a heterotetramer complex comprising two light (L) chains and two heavy (H) chains. In some embodiments, the two light chains and the two heavy chains are identical. In some embodiments, the two light chains comprise different amino acid sequences. In some embodiments, the two heavy chains comprise different amino acid sequences. In some embodiments, the antibody is a full-length antibody (e.g., comprising two full-length light chains and two full-length heavy chains). In some embodiments, the antibody is an antibody fragment comprising a part of the constant region of the heavy chain containing an engineered mutation, such as Fab, F(ab')2, Fab'-SH, Fv, scFv and a single heavy chain antibody. In certain embodiments, the antibody is a mammalian antibody (such as a human antibody, a non-human primate antibody, a mouse antibody, a rat antibody, etc.). In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a multispecific antibody.

[0127] Multispecific Antibodies

[0128] Multispecific antibodies have binding specificity (e.g., two, three or more binding specificities) for more than one antigen. In some embodiments, the antibody quantified using the method provided herein is a bispecific antibody. In some embodiments, the bispecific antibody comprises two different binding specificities to the same antigen (e.g., different binding affinities and / or specific epitopes with the same antigen). In some embodiments, the bispecific antibody comprises binding specificity to two different antigens. In some embodiments, the bispecific antibody is a full-length or complete antibody. It is contemplated that the method provided herein is used together with bispecific or multispecific antibody formats known in the art. Exemplary bispecific and multispecific antibody formats include, but are not limited to, those described below.

[0129] For example, "protrusion into a hole" is a design strategy for engineering antibody heavy chain homodimers for heterodimerization (e.g., for the production of bispecific antibodies, multispecific antibodies, or one-armed antibodies). Typically, such techniques involve introducing a protrusion ("protrusion") at the interface of a first polypeptide (e.g., the first CH3 domain in the first antibody heavy chain) and a corresponding cavity ("hole") in the interface of a second polypeptide (e.g., the second CH3 domain in the second antibody heavy chain), so that the protrusion can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. Protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., the first CH3 domain in the first antibody heavy chain) with larger side chains (e.g., arginine, phenylalanine, tyrosine, or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine, serine, valine, or threonine), a compensatory cavity of the same or similar size as the protrusion is generated in the interface of the second polypeptide (e.g., the second CH3 domain in the second antibody heavy chain). See, e.g., U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; and 8,679,785, each of which is incorporated herein by reference in its entirety. Exemplary protuberance-entry mutation groups include, but are not limited to, those shown in Table 1 below:

[0130] Table 1. Exemplary protuberance-entry mutation groups

[0131]

[0132] WO 2011 / 131746 describes a bispecific antibody comprising an asymmetric mutation at one of positions 366, 368, 370, 399, 405 and 407 (according to EU numbering) in each CH3 domain. After incubation under reducing conditions, the mutation drives a directed "Fab arm" or "half molecule" exchange between two monospecific IgG1, IgG4 or IgG4-like antibodies.

[0133] US2009 / 0232811 and Schaefer et al. (2011) PNAS USA. 108(27): 11187-11192 describe Crossmab technology, i.e., a bispecific antibody format that involves exchanging one or more heavy and light chain domains within the antigen-binding fragment (Fab) of half of the bispecific antibody. The correct association of the light chain and its cognate heavy chain is achieved by exchanging the heavy and light chain domains in the antigen-binding fragment (Fab) of half of the bispecific antibody. This "crossing" retains antigen binding affinity, but makes the two arms so different that light chain mispairing no longer occurs. Exemplary Crossmab formats include Crossmab Fab(i.e., where the CL and VL domains are exchanged with the CH1 and VH domains, respectively), Crossmab VH-VL (i.e., where the VL and VH domains are swapped) and Crossmab CH1-VCL (ie, wherein the CH1 and CL domains are swapped). See also WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253 and WO 2009 / 080254, the contents of each of which are incorporated herein by reference in their entirety.

[0134] WO 2007 / 147901 describes a strategy for engineering antibody heavy chains to heterodimerize (e.g., for producing bispecific antibodies, multispecific antibodies or one-armed antibodies), which requires the introduction of asymmetric mutations in the Fc domain (i.e., K253E, D282K, K322D in the first Fc domain, and D239K, E240K and K292D in the second Fc domain) and the introduction of asymmetric mutations in the CH / CL domain (i.e., K96E in CH1 and E15K in CL). It is reported that such asymmetric mutations both destroy the ionic interactions that stabilize the homodimerization of the half antibody and promote the heterodimerization of the Fc domain.

[0135] WO 2009 / 089004 describes heteromultimeric proteins (e.g., bispecific, multispecific or one-armed antibodies) comprising asymmetric pairs of mutations in the CH3-CH3 interface that electrostatically disfavor Fc homodimerization but electrostatically favor Fc heterodimerization. See, e.g., Tables 2a and 2b in WO 2009 / 089004).

[0136] Several antibody-like proteins with CODV (cross dual variable) are described in Steinmetz et al. (2016) MABS. 8(5):867-878, WO 2012 / 135345, WO2016 / 116626 and U.S. Pat. Nos. 9,181,349 and 9,221,917, each of which is incorporated herein by reference in its entirety. The CODV structure produces a circular, self-contained structure that acts as a self-supporting framework, maintaining the parent antibody affinity for the two antigens. The CODV antibody-like protein may be (a) bivalent and / or bispecific; (b) trivalent and / or trispecific; (c) trivalent and / or bispecific; or (d) tetravalent and / or bispecific. In an exemplary form, the polypeptide comprises two polypeptide chains having a structure represented by the following formula: V L1 -L1-V L2 -L2-C L , and two polypeptide chains having structures represented by the following formula: V H2 -L3-V H1 -L4-CH1 -Fc, where V L1 is the first immunoglobulin light chain variable domain; V L2 V is the second immunoglobulin light chain variable domain; H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 Immunoglobulin C H1 Heavy chain constant domain; Fc contains the immunoglobulin hinge region and C H2 , C H3 An immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. In another exemplary form, the polypeptide comprises two polypeptide chains forming two antigen binding sites, wherein the first polypeptide has a structure represented by the following formula: V L1 -L1-V L2 -L2-C L -Fc, and the second polypeptide chain has a structure represented by the following formula: H2 -L3-V HI -L4-C H1 -Fc, where: V L1 is the first immunoglobulin light chain variable domain; V L2 V is the second immunoglobulin light chain variable domain; H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 is the constant domain of the immunoglobulin CH1 heavy chain; C H2 Immunoglobulin C H2 Heavy chain constant domain; C H3 Immunoglobulin C H3 Heavy chain constant domain; Fc contains the immunoglobulin hinge region and C H2 , C H3 immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; wherein the first and second polypeptides form a crossover light chain-heavy chain pair. In a third exemplary form, the polypeptide comprises three polypeptide chains that form two antigen binding sites, wherein the first polypeptide chain has a structure represented by the following formula: L1 -L1-V L2 -L2-C L , wherein the second polypeptide chain has a structure represented by the following formula: V H2 -L3-V HI -L4-C H1 -Fc, the third polypeptide chain comprises an antibody Fc region, wherein: VL1 is the first immunoglobulin light chain variable domain; V L2 V is the second immunoglobulin light chain variable domain; H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 Immunoglobulin C H1 Heavy chain constant domain; C H2 Immunoglobulin C H2 Heavy chain constant domain; C H3 Immunoglobulin C H3 Heavy chain constant domain; Fc contains the immunoglobulin hinge region and C H2 , C H3 immunoglobulin heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the first and second polypeptides form a crossover light chain-heavy chain pair. In a fourth exemplary form, the polypeptide comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a structure represented by the following formula: V L1 -L1-V L2 -L2-C L , and the second polypeptide chain comprises a structure represented by the following formula: V H2 -L3-V H1 -L4-C H1 , where: V L1 is the first immunoglobulin light chain variable domain; V L2 V is the second immunoglobulin light chain variable domain; H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 Immunoglobulin C H1 heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

[0137] Wu et al. (2007) Nat Biotechnol. 25: 1290-7 first described a bispecific tetravalent immunoglobulin called a tetravalent bispecific tandem immunoglobulin (TBTI) or a dual variable domain immunoglobulin (DVD-Ig). Like conventional IgG, TBTI-DVD-Ig contains two heavy chains and two light chains. However, both the heavy and light chains of TBTI-DVD-Ig contain additional variable domains, which are connected to the N-termini of the VH and VL of existing monoclonal antibodies through flexible, naturally occurring linker sequences. Therefore, when the heavy and light chains are combined, the resulting TBTI-DVD-Ig contains four antigen recognition sites. See also U.S. Patent Nos. 9,029,508; 9,109,026; 9,035,027; 9,046,513, 8,388,965; 9,732,162; 9,738,728; European Patent No. 2573121 Bl, and WO2012 / 135345, the contents of each of which are incorporated herein by reference in their entirety.

[0138] US2017 / 00320967 and WO 2017 / 180913 (the contents of which are expressly incorporated herein by reference in their entirety) describe binding proteins (such as trivalent and / or trispecific antibodies) comprising four polypeptide chains forming three antigen binding sites. In some embodiments, the binding protein is trivalent. In some embodiments, the binding protein is trispecific. In an exemplary form, the first polypeptide chain comprises a structure represented by the following formula: V L2 -L1-V L1 -L2-C L ; The second polypeptide chain comprises a structure represented by the following formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 ; The third polypeptide chain comprises a structure represented by the following formula: V H3 -C H1 -Hinge-C H2 -C H3 ; The fourth polypeptide chain comprises a structure represented by the following formula: V L3 -C L , where V L1 is the first immunoglobulin light chain variable domain; V L2 V is the second immunoglobulin light chain variable domain; L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; C H1 Immunoglobulin C H1 a heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the first and second polypeptides form a crossover light chain-heavy chain pair; and wherein the second polypeptide chain or the third polypeptide chain comprises the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8) or GSFFLVSKLTVD (SEQ ID NO: 9).

[0139] In some embodiments, the length of the linker L1, L2, L3 and L4 ranges from no amino acid (length = 0) to about 100 amino acids, or less than 100, 50, 40, 30, 20 or 15 amino acids or less. The length of the linker can also be 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid. L1, L2, L3 and L4 in a binding protein can all have the same amino acid sequence, or can all have different amino acid sequences.

[0140] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO:21); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO:22); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:23); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:24); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:25). Other combinations of amino acid residues may be used, such as the peptide Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:26), the peptide Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:27), and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:28). Other suitable linkers include single Ser and Val residues; the dipeptides Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO:29), Thr-Val-Ala-Ala-Pro (SEQ ID NO:30), Gln-Pro-Lys-Ala-Ala (SEQ ID NO:39), Gln-Arg-Ile-Glu-Gly (SEQ ID NO:31); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO:37), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO:32), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO:33), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO:34). The examples listed above are not intended to limit the scope of the present disclosure in any way, and linkers comprising randomly selected amino acids selected from valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine and proline have been shown to be suitable for binding proteins.

[0141] The identity and sequence of the amino acid residues in the joint can vary according to the type of secondary structure element required to be achieved in the joint. For example, glycine, serine and alanine are most suitable for joints with maximum flexibility. If a more rigid and extended joint is desired, some combination of glycine, proline, threonine and serine is useful. Depending on the desired properties, any amino acid residue can be considered as a joint combined with other amino acid residues to build a larger peptide joint as required.

[0142] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is 3 to 12 amino acid residues, the length of L2 is 3 to 14 amino acid residues, the length of L3 is 1 to 8 amino acid residues, and the length of L4 is 1 to 3 amino acid residues. In some embodiments, the length of L1 is 5 to 10 amino acid residues, the length of L2 is 5 to 8 amino acid residues, the length of L3 is 1 to 5 amino acid residues, and the length of L4 is 1 to 2 amino acid residues. In some embodiments, the length of L1 is 7 amino acid residues, the length of L2 is 5 amino acid residues, the length of L3 is 1 amino acid residue, and the length of L4 is 2 amino acid residues.

[0143] In some embodiments, L1, L2, L3 and / or L4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 35). In some embodiments, L1 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 35). In some embodiments, L3 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 35).

[0144] In some embodiments, L1, L2, L3 and / or L4 comprise the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 36), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). In some embodiments, L1 comprises the sequence Ser, L2 comprises the sequence Arg-Thr, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33), and L4 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 36).

[0145] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13; the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15. (SEQ ID NOs: 12, 13, 14, and 15 correspond to SEQ ID NOs: 3, 4, 1, and 2 disclosed in WO 2017 / 074878, respectively). The amino acid sequences of additional exemplary trispecific antibodies are described in WO 2017 / 074878, the contents of which are incorporated herein by reference in their entirety.

[0146] The chain exchange engineered domain (SEED) platform designed for the generation of asymmetric and bispecific antibody-like molecules is based on exchanging structurally related sequences of immunoglobulins in a conserved CH3 domain. The alternating sequences of human IgA and IgG in the SEED CH3 domain produce two asymmetric but complementary domains, called AG and GA. The SEED design allows efficient generation of AG / GA heterodimers and is not conducive to the formation of AG / AG and GA / GA homodimers. See, for example, Muda et al. (2011) Prot Engineering, Design & Selection. 24 (5): 447-454 and U.S. Patent No. 8,871,912, the contents of which are incorporated herein by reference in their entirety.

[0147] Other bispecific and multispecific antibody formats are described in Klein et al. (2012) mAbs 4:6, 653-663; Spiess et al. (2015) "Alternative molecular formats and therapeutic applications for bispecific antibodies." Mol. Immunol. 67(2Pt A):95-106; Egan et al. (2017) mAbs. 9(1):68-84; Liu et al. (2017) Front Immunol. 8:38; and Weidle et al. (2013) Cancer Genomics Proteomics. 10(1):1-18.

[0148] Target Antigen

[0149] In certain embodiments, the polypeptide to be quantified according to the methods provided herein (i.e., a polypeptide comprising a portion of an antibody heavy chain constant region containing an engineered mutation) specifically binds to a target antigen. Exemplary target antigens include, but are not limited to, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL 11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,IgE receptor), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), ), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IG F1R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, class II MHC, NCR3LG1, NK G2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigen targets are human antigen targets. In embodiments where the polypeptide comprising an antibody heavy chain constant region comprising an engineered mutation is a bispecific or multispecific target binding protein, the target antigen may be any two (or more) of the exemplary antigens listed above.

[0150] Conjugate

[0151] In some embodiments, the polypeptide to be quantified according to the method provided herein (i.e., a polypeptide comprising an antibody heavy chain constant region containing an engineered mutation) is conjugated with a drug such as a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or a fragment thereof of a bacterial, fungal, plant or animal origin). Exemplary drugs include, but are not limited to, daunomycin, doxorubicin, methotrexate, vindesine, BCNU, streptozotocin, vincristine and 5-fluorouracil. Exemplary toxins include, but are not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, geldanamycin, maytansine (or other maytansine-like compounds), auristatin, dolastatin, calicheamicin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, and CC1065. Cytotoxic agents and linkers for conjugating agents to antibodies are known in the art; see, e.g., Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

[0152] In some embodiments, the polypeptide to be quantified according to the methods provided herein (i.e., a polypeptide comprising an antibody heavy chain constant region containing an engineered mutation) is conjugated to a detectable portion capable of directly or indirectly producing a detectable signal. In certain embodiments, the detectable marker is a radionuclide. A variety of radionuclides can be used to produce radioconjugated polypeptides for clinical and research purposes. Examples include 13 C. 15 N. 17 O. 19 F. 32 P. 90 Y. 99m Tc, 111 In, 123 I. 125 I. 131 I. 131 In, 153 Sm, 186 Re, 188 Re, 211 At 212 Bi, 212 Pb and radioactive isotopes of Lu, Mn, Fe and Gd. In certain embodiments, the detectable label is a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase.

[0153] The foregoing written description is considered sufficient to enable those skilled in the art to practice the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, in addition to those shown and described herein, various modifications will become apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims.

[0154] Example

[0155] Example 1: Identification of surrogate peptides for use in a universal mass spectrometry-based assay to detect polypeptides comprising antibody heavy chain constant regions in a sample

[0156] The following selection criteria were applied to identify alternative peptides for detection of therapeutic antibodies in biological samples via mass spectrometry:

[0157] ●6-26 amino acids in length;

[0158] ●No adjacent endopeptidase cleavage site;

[0159] ● There is no methionine (M) or cysteine ​​(C) in the peptide sequence;

[0160] ● There is no asparagine followed by glycine or serine in the peptide sequence (i.e., NG or NS);

[0161] A constant region derived from an antibody heavy chain; and

[0162] ● Contains at least one engineered substitution mutation (ie, excludes naturally occurring polymorphisms).

[0163] First, the Fc sequences of three exemplary therapeutic antibodies were aligned. Figure 1 . SEQ ID NO:1 is a portion of an anti-target A antibody heavy chain. SEQ ID NO:1 does not contain engineered mutations. SEQ ID NO:2 is a portion of the first chain (i.e., "Chain 1") of a trispecific construct (i.e., "TRI-1"). TRI-1-Chain 1 comprises an antibody heavy chain comprising S354C and T366W mutations (i.e., "knob" mutations) and M428L / N434S mutations (which extend the in vivo half-life of TRI-1). SEQID NO:3 is a portion of the second chain (i.e., "Chain 2") of TR1. TRI-1-Chain 2 comprises an antibody heavy chain comprising Y349C / T366S / 368A / 407V "hole" mutations and M428L / N434S mutations. SEQ ID NO:4 is a portion of the first chain (i.e., "Chain 1") of a second trispecific construct (i.e., "TRI-2"). TRI-2-chain 1 comprises an antibody heavy chain comprising Y349C / T366S / 368A / 407V mutations (i.e., "hole" mutations) and M428L / N434S mutations. SEQ ID NO: 5 is a portion of the second chain (i.e., "chain 2") of TR2. TRI-2-chain 2 comprises a heavy chain comprising S354C / T366W "knob" mutations and M428L / N434S mutations. All amino acid positions discussed above are numbered according to the EU numbering scheme.

[0164] Candidate peptides containing at least one engineered substitution mutation are underlined and are predicted to be Figure 1 The peptide TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) met all the criteria listed above.

[0165] Next, a BLAST search was performed using TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) (i.e., "engineered TTPP peptide") as a query sequence against mouse, cynomolgus monkey, and human protein databases to determine whether such a sequence is found in any native proteins in these species. The only sequence that exhibited 100% amino acid identity with the engineered TTPP peptide was a known protuberance-entry antibody. The other sequences that exhibited 94% amino acid identity with the engineered TTPP peptide corresponded to human Fc domains containing Y407 substitution mutations other than Y407V and endogenous human Fc domains.

[0166] The experiments described below were performed to evaluate the feasibility of trypsin digestion of the trispecific construct TRI-1 to generate engineered TTPP peptides. Briefly, SMART TM Digest kit (ThermoFisher) digested 10 μg of TRI-1 (which contained Figure 1 SEQ ID NOs: 2 and 3 shown). The digestion reaction solution was incubated at 70°C while shaking at 1400 rpm for 75 minutes. The digested sample was then centrifuged at 1000 x g for 2 minutes, and the supernatant was collected for purification and concentration.

[0167] The desired number of wells in the SOLAμHRP solid phase extraction (SPE) plate (ThermoFisher) was balanced with 200 μL of acetonitrile and then adjusted with 200 μL of 0.1% trifluoroacetic acid (TFA) aqueous solution. Then an additional 400 μL of 0.1% TFA aqueous solution was added to each well. The sample supernatant was added to the well and passed through. The wells were then washed with 500 μL of 0.1% TFA aqueous solution and then with 500 μL of 0.1% formic acid aqueous solution. The digested sample was eluted with 2x 25 μL of 80% acetonitrile aqueous solution with 0.1% formic acid. The eluate was diluted with 50 μL of 0.1% formic acid aqueous solution and vortexed briefly at 400 rpm.

[0168] 50 μL of each digested sample was analyzed using a Waters H-Class Acquity UPLC coupled to a ThermoQ Exactive mass spectrometer operating under typical peptide mapping conditions.

[0169] like Figures 2A-2C As shown, trypsin digestion of TRI-1 reproducibly produces engineered TTPP peptides that are easily detected and fragmented. Figure 2A The extracted ion chromatogram for m / z 905.40-907.60 is shown in Figure 1, which is a subset of the natural isotopic range of engineered TTPP. Engineered TTPP elutes at approximately 19 minutes. In the total ion chromatogram ( Figure 2B In addition, the engineered TTPP showed good fragmentation for MS / MS analysis ( Figure 2C ).

[0170] Example 2: Detection of TTPP peptide in serum

[0171] Next, a series of experiments were performed to determine whether the engineered TTPP peptides could be detected in human, monkey, and mouse sera by LC-MS / MS without significant background interference. Mouse and cynomolgus monkey sera were tested because these animals are commonly used in preclinical therapeutic antibody studies.

[0172] In brief, 100 μL of vortexed G protein magnetic beads (Pierce) and 100 μL of PBST were added to the wells of a 96-well microplate (Qiagen). The plate was gently shaken to ensure a uniform mixture. The 96-well plate was placed on an Alpaqua 96S SuperMagnet 96-well magnet for 1 minute, then placed on a Qiagen A-type 96-well magnet for 1 minute, and then the solution was removed. The wash cycle was repeated 3 more times with 200 μL of PBST. After washing, 140 μL of PBST was added to each well containing beads. Then, 30 μL of (1) mouse serum, (2) monkey serum, (3) human serum, or (4) 2 μg / mL TRI-1 was added to the wells. 30 μL of calibrators, QCs, and samples were added to separate wells containing beads, PBST, and serum. The 96-well plate was then covered and shaken at 600 RPM for 1 hour and washed four times. Calibrators, QCs, and samples were eluted by adding 90 μL of 0.1% TFA to each well and gently vortexing the plate to ensure a uniform mixture. The plate was placed on an Alpaqua 96S Super Magnet 96-well magnet for 1 minute and then on a Qiagen Type A 96-well magnet for 1 minute. The eluate was then transferred to a 2.0 mL 96-well deep well Protein LoBind plate (Eppendorf). The elution procedure was then repeated. The pH of the eluate was neutralized with 20 μL of 1M tris-HCl pH 8 to a total volume of 200 μL. Simply, 200 μL of SMART digestion buffer was added to each eluate. The samples were incubated at 70°C with shaking at 400 rpm for 75 minutes and then centrifuged at 1000 x g for 2 minutes to precipitate the SMART digestion resin.

[0173] The wells in the SOLAμHRP SPE plate were equilibrated with 200 μL of acetonitrile and then adjusted with 200 μL of 0.1% TFA in water. An additional 400 μL of 0.1% TFA in water was added to each well. Each sample supernatant was added to a separate well and passed through. The wells were washed with 500 μL of 0.1% TFA in water and then 500 μL of 0.1% formic acid in water. The digested samples were eluted with 2x 25 μL of 80% acetonitrile in water with 0.1% formic acid. The eluate was diluted with 50 μL of 0.1% formic acid in water and vortexed briefly at 400 rpm.

[0174] SMART digests of the samples were analyzed using a Waters H-Class Acquity UPLC and a ThermoQ Exactive mass spectrometer under standard peptide mapping conditions.

[0175] Engineered TTPP peptide (m / z 905.47 2+ ) is shown in the extracted ion chromatogram Figures 3A-3C The extracted ion chromatogram of the engineered TTPP peptide in TRI-1 digest (2 μg / mL in PBST) is shown in FIG. Figure 3D The peak at 20.65 minutes in the TRI-1 digest was not present in any of the serum backgrounds tested.

[0176] To confirm that the engineered TTPP peptides could be detected in the serum background, mouse, monkey, and human serum samples were spiked / fortified with 20 μg / mL TRI-1 and subjected to antibody pull-down, digestion, and mass spectrometry analysis as described above.

[0177] Engineered TTPP peptide (m / z 905.47 2+ ) is shown in the extracted ion chromatogram Figures 4A-4C As shown, while TRI-1 digestion in PBST was as shown Figure 4D After TRI-1 was spiked into each serum background, the engineered TTPP peptide was clearly seen in all backgrounds.

[0178] Prepare serial dilutions of TRI-1 into an appropriate serum background to give final concentrations of 0.2, 2, and 20 μg / mL to determine if the signal increases proportionally to the amount of analyte in the sample. An aliquot of serum without antibody spike-in was used as a background control. Pulldowns, digestion, SPE cleanup, and LC-MS / MS were performed as described above.

[0179] The extracted ion chromatograms of engineered TTPP, unlabeled FNWY, and FNWY(heavy) derived from the analysis of three tested concentrations of TRI-1 in PBST (20, 2, and 0.2 μg / mL) are shown in Figure 2 , respectively. Figures 5A-5C , 6A-6C and 7A-7C. The extracted ion chromatograms of engineered TTPP, unlabeled FNWY and FNWY (heavy) derived from the analysis of four tested concentrations of TRI-1 in mouse serum (20, 2, 0.2 and 0 μg / mL) are shown in Figures 8A-8C , 9A-9C and 10A-10C. The extracted ion chromatograms of engineered TTPP, unlabeled FNWY and FNWY (heavy) derived from the analysis of four tested concentrations of TRI-1 in monkey serum (20, 2, 0.2 and 0 μg / mL) are shown in Figures 11A-11C, 12A-12C and 13A-13C. The extracted ion chromatograms of the engineered TTPP derived from the analysis of four tested concentrations of TRI-1 in human serum (20, 2, 0.2 and 0 μg / mL) are shown in Figures 14A-14C Shown in.

[0180] The peak areas of the engineered TTPP peptide and the heavy isotope peptide FNWYVDGVEVHNAK (SEQ ID NO: 10) were determined. See Table 2 below. FNWY (heavy) is derived from a stable isotope labeled universal monoclonal antibody (SILU TM MAB) digested internal standard.

[0181] Table 2. Summary of the results of quantitative assays

[0182]

[0183]

[0184] The area ratios of engineered TTPP to FNWY (heavy) in the PBST, mouse serum, and monkey serum backgrounds are also shown in Table 2 and are presented in Fig.15A -D are plotted against input TRI-1 concentration. The area ratios of engineered FNWY of PBST to FNWY (heavy) are shown in Table 2 and Fig.15A , 15C and 15D are plotted relative to input antibody concentration.

[0185] The above data demonstrate that there is little to no background interference from the TTPP peptide in mouse serum, monkey serum, or human serum, and that the signal generated by the TTPP peptide correlates with the amount of peptide in the sample.

[0186] Table 3. Amino acid sequences in Examples 1 and 2

[0187]

[0188]

[0189] Example 3: Use of TTPP surrogate peptides for detection and quantification of exemplary trispecific antibodies in a universal mass spectrometry-based assay.

[0190] Overview

[0191] Therapeutic antibodies are attractive tools for targeting diseases and their market is growing rapidly. Ligand binding assays are the standard method for measuring therapeutic antibodies. However, to provide support for their development, new bioanalytical strategies are necessary. LC-MS / MS represents a complementary approach that provides the specificity and higher reproducibility required for good laboratory practice (GLP) studies. Moreover, LC-MS / MS overcomes some of the limitations associated with ligand binding assays (LBAs), such as matrix interferences (e.g., components in the sample that may hinder accurate quantification of therapeutic antibodies) and the development of reagents, which can be expensive and time-consuming.

[0192] IgG represents most therapeutic proteins clinically at present.In order to reduce immunogenicity, therapeutic antibodies are engineered to include human antibody constant regions, such as human IgG constant regions.Due to being difficult to select suitable alternative peptides that can be used to distinguish endogenous human IgG from therapeutic IgG antibodies in samples (such as plasma, serum or blood samples) without the help of immunoaffinity steps, it is always challenging to develop bioanalysis LC-MS assays to quantitatively measure such therapeutic antibodies.In order to overcome this problem, the constant region of exemplary therapeutic antibodies is engineered to include substitution mutations, for example, substitution mutations found in non-natural antibody constant regions of human antibody constant regions or non-human primates or preclinical animals (such as mice).This strategy allows accurate quantification of therapeutic antibodies in non-human samples and human samples without matrix interference.

[0193] The following describes an LC-MS / MS assay that does not require affinity extraction and can rapidly and accurately quantify trivalent, trispecific antibodies against soluble targets in plasma. By avoiding the immunoaffinity step, this method will allow accurate quantification of total drug (the sum of target-bound drug plus free drug). Moreover, because the substitution mutations are in the antibody constant region, this method can be used to quantify any therapeutic protein containing an antibody constant region.

[0194] Analytical procedures

[0195] The surrogate peptides selected for quantification of an exemplary trispecific antibody by LC-MS / MS are shown in Fig.16 and 17 and in Table 4 below. These alternative peptides were selected based on their linear response. Peptides whose sequences were not found in human proteins or proteins of non-human primates were used (i.e., SEQ ID NOs: 7, 16, and 38). For absolute quantification, the engineered peptide TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) was used (see Fig.16 and 17and Table 4 below). To ensure the integrity of the analytes, the ratio between the areas under the peaks of TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) and the other peptides listed in Table 4 was monitored in samples quantified by LC-MS / MS. The ratio remained constant at all time points (results not shown).

[0196] Table 4. List of surrogate peptides and their MS parameters for detection

[0197]

[0198] A stable isotope-labeled version of TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) was used as an internal standard ( Fig.19B ).

[0199] A rapid and robust workflow has been developed to support preclinical studies (see Fig.18 ). The method is based on the precipitation digestion protocol described in Ouyang et al. (2012) Bioanalysis. 4(1), 17-28. Each step of the digestion protocol was optimized to maximize the recovery of trispecific antibodies from the sample and the detection of surrogate peptides. (The workflow for samples from human subjects included a protein A immunopurification step before the denaturation step).

[0200] The method uses only 10 μL of plasma sample. Since a small amount of sample is sufficient, the alternative peptide quantification method is suitable for measuring preclinical samples from, for example, rodents. The standard digestion time was also optimized. The best signal intensity was observed when the samples were digested for 2 hours at 37°C. To further improve the sensitivity of the quantitative method, the samples were subjected to solid phase extraction (SPE). The best recovery was obtained using the OASIS MCX box.

[0201] Validation in rat plasma

[0202] Method validation was performed in rat plasma to support GLP toxicology studies. A quadratic regression model was selected with an average regression coefficient of 0.979 over three independent calibration curves with three replicate runs and eight nominal concentrations ( Fig.19A ). The variability of the analysis was assessed by measuring the accuracy and precision of quality control (QC) samples at LLOQ (lowest limit of quantitation), low, intermediate and high of three independent preparations. Table 5 summarizes the accuracy and precision within / between runs. Matrix effects were performed using six different batches of plasma (see Table 6 below). The stability of the antibodies in plasma was evaluated in adulterated / spiked human plasma stored at -80°C and at 37°C for 1-3 and 6 months. No degradation was observed during these time intervals.

[0203] Table 5. Inter-run and intra-run accuracy and precision for three independent samples at LLOQ, low, mid, and high

[0204]

[0205] Table 6. Matrix effects of six independent batches of rat plasma

[0206]

[0207] Additional experiments were performed to determine whether the trispecific antibody could be detected at 5 μg / ml using the surrogate TTPP peptide (SEQ ID NO: 7) when spiked into monkey and human plasma. Figures 22A-22F As shown, in adulterated / spiked rat plasma ( Fig. 22B ), adulterated / spiked monkey plasma ( Fig.22D ) and adulterated / spiked human plasma ( Fig.22F ) but not in blank rat plasma ( Fig.22A )、blank monkey plasma( Fig. 22C ) or blank human plasma ( Fig.22E ) was not detected.

[0208] As discussed elsewhere herein, ligand binding assays (LBAs), such as ELISAs, have traditionally been used to detect and quantify therapeutic proteins (e.g., monoclonal antibodies) in biological samples. Quantification of monoclonal antibodies via LBAs typically involves the use of a target antigen as a capture agent or detector. However, if LBAs are used to detect trispecific antibodies and soluble targets of trispecific antibodies, inaccurate results may be produced. For trispecific antibodies, quantification by LBA would require the development of three different assays (i.e., one assay for each target antigen), which would produce three different concentration data sets, which is unmanageable for preclinical species or human PK exposure assessments. Moreover, it is difficult to distinguish between the amount of free antibody and target-bound antibody and total antibody in a sample using LBAs. In this example, a universal LC-MS assay that can be used in preclinical and clinical stages is shown to be able to measure trispecific antibodies against soluble targets with high accuracy. As demonstrated by the absence of interfering signals in blank plasma samples, the assay is shown to be specific (see Figures 22A-22F As shown in Table 5, this assay was also shown to be reproducible, with accuracy % difference estimates and inter- and intra-run % precision all within international regulatory acceptance criteria defined by FDA and EMA guidelines for bioanalytical method validation (i.e., % accuracy no greater than ±20%, % precision no greater than ±20%).

[0209] Materials and methods for preclinical species

[0210] The trispecific antibodies were provided as stock solutions at a nominal concentration of 25 mg / mL in 10 mM histidine, 8% sucrose, 0.04% polysorbate 80, pH 6.0. Calibration standards and QCs were obtained by diluting the stock solutions in rat plasma.

[0211] The precipitation digestion of rat plasma containing trispecific antibodies was optimized according to the procedure previously described in Ouyang et al. (2012) Bioanalysis.4(1), 17-28. Briefly, 10 μl of each rat plasma sample containing trispecific antibodies was placed in a 1.5 mL Lobind tube and mixed with an equal volume of PBS buffer at pH 7.4. Plasma proteins and therapeutic antibodies in each sample were precipitated by adding 30 μL of methanol to the tube. After vortexing, the samples were centrifuged at 2000 rcf (relative centrifugal force) at 5 ° C for 6 minutes. The supernatant was discarded and the protein precipitate was resuspended in 40 μl of 200 mM ammonium bicarbonate buffer (ABC). 5 μl of 1 M dithiothreitol (DTT) was added to each resuspended precipitate and incubated at 56 ° C for 30 minutes under slow stirring. The samples were then cooled at room temperature for 10 minutes. Next, 10 μl of internal standard working solution (100 μg / mL) was added to each sample and mixed. The samples were further incubated with 10 μL of iodoacetamide (IAA) (100 mM) in 100 mM ABC buffer at room temperature for 30 minutes under gentle rotation while protected from light. 30 μg / mL of trypsin and 100 μL of ABC buffer were added to each sample, and then the samples were incubated for 2 hours at room temperature under gentle rotation.

[0212] The digestion reaction was terminated with 30 μL 10% formic acid (FA), and centrifuged at 16000rcf for 10 minutes at 5 ° C. Using a positive pressure processor manifold, the supernatant was subjected to solid phase extraction (SPE) with OASIS MCX elution plates. In brief, the plate was balanced with 200 μL of methanol, and then balanced with 200 μL of 0.5% FA. Each supernatant of 200 μL was applied to the plate, and washed with 20% MeOH solution of 0.5% FA, 80% MeOH solution of 0.5% FA of 200 μL, and 20% MeOH solution of 0.5% FA of 200 μL. Finally, the ammonium hydroxide buffer 5% / methanol (40:60, v / v) of 2x25 μL was used to elute the sample. The extract of 10 μL was diluted in 200 μL of water / formic acid (100:0.5, v / v). Each eluted sample of 10 μL was used for LC-MS / MS analysis.

[0213] LC-MS / MS analysis was performed using The analysis was performed on a SCIEX triple quadrupole API5500 mass spectrometer with software version 1.6.2. The system was connected to an AGILENT 1290INFINITY liquid chromatography system. An X-BridgeProtein BEH C4 column was used; (Waters 100 x 2.1 mm ID, 3.5 μm particle size) Chromatography was performed at 50° with a gradient of solvent A (0.1% FA) and B (0.1% FA in acetonitrile). After 2.5 min equilibration with solvent A, the aqueous solution was adjusted to 55% after 5 min at a flow rate of 600 μL / min.

[0214] MS data were collected in positive mode with an ion spray voltage of 5500 V, a source temperature of 600° C., and a desolvation temperature of 60° C. CE energies were optimized for each peptide (see Table 4).

[0215] Materials and methods for human samples

[0216] Using Agilent Affinity purification was performed on the BRAVO liquid handling platform as follows: In a 96-well plate, 90 μl of PBST and 10 μl of human plasma sample were added sequentially. The plate was vortexed for 5 minutes and placed on the BRAVO platform in position 4 maintained at approximately 20°C. The 96-well plate was filled with 20 μl of 1 M Tris and the following solutions were filled into the reservoirs in sequential order: perfusion and equilibration buffer (phosphate-buffered saline (PBST)), cartridge wash 1 (1 M NaCl in PBST), cartridge wash 2 (PBST), and elution and syringe wash buffer (1% formic acid). Next, run Affinity purification protocol on the BRAVO liquid handling platform.

[0217] Denaturation, reduction, alkylation and digestion were performed as follows: Centrifuge the 96-well plate in position 9 for 30 seconds. Sequentially add 50 μl of 0.2% RAPIGEST in 50 mM ammonium bicarbonate. TM Surfactant and 20 μL of 100 mM DL-dithiothreitol in 100 mM ammonium bicarbonate. Next, the plate was centrifuged for 30 seconds and incubated at about 56 °C for 30 minutes with gentle agitation. The samples were cooled at room temperature. 20 μL of 250 mM iodoacetamide in 50 mM ammonium bicarbonate and 50 μl of 200 mM ammonium bicarbonate @ 0.01% were added. 20 ISW. The plate was centrifuged for 30 seconds and then incubated for 30 minutes at about 20°C with gentle agitation in the dark. 100 μL of 1 μg / μL fresh trypsin in 50 mM ammonium bicarbonate was added and the sample was incubated overnight at about 37°C. 20 μL of 10% formic acid in water was then added (to stop digestion).

[0218] Solid phase extraction using Oasis PRIME MCXμ elution plates was performed as follows: 200 μL of each supernatant was applied to the plate and washed with 200 μL 0.5% FA and 200 μL 0.5% FA in 80% MeOH. The sample was finally eluted with 50 μL of ammonium hydroxide buffer 5% / methanol (40:60, v / v) and 150 μL of water containing 0.5% formic acid was added to the eluted sample. The plate was loaded into the autosampler tray maintained at +10°C and 10 μL of each sample was injected.

[0219] Human plasma samples were subjected to LC-MS / MS analysis as described above for preclinical samples.

[0220] Table 7. Amino acid sequences in Example 3

[0221]

[0222]

[0223] Example 4: Proof of concept study using TTPP surrogate peptides to detect and quantify exemplary trispecific antibodies following administration to mice in a universal mass spectrometry-based assay

[0224] Materials / Methods

[0225] The materials used in this example are: trispecific antibody, serum (mouse), protein G resin tip (Agilent), C18 resin tip (Agilent), SILU TM MAB stable isotope labeled universal monoclonal antibody internal standard (Sigma), heavy labeled engineered peptide internal standard (Thermo), 50 mM ammonium bicarbonate, 8 M urea with 550 mM Tris base pH 8, 1 M sodium chloride in phosphate buffered saline, 125 mM dithiothreitol, 250 mM iodoacetamide, phosphate buffered saline (PBS), trypsin (Promega), LysC (Promega), water, acetic acid, trifluoroacetic acid (TFA), formic acid, acetonitrile

[0226] Calibration curve preparation:

[0227] A calibration curve was prepared by serially diluting the trispecific antibody in mouse serum. The concentrations tested in this proof of concept study were 0.012, 0.024, 0.049, 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, and 800 μg / mL. Serum without the trispecific antibody was used as the zero point.

[0228] Stable isotope labeled internal standard preparation:

[0229] On the day of use, add 500 μL of 0.1% formic acid to the SILU TM MAB stable isotope labeled universal monoclonal antibody (Sigma Aldrich) was placed in 100 μg vials and allowed to stand at room temperature according to the manufacturer's instructions. The antibody concentration was 0.2 μg / L.

[0230] Heavy labeled engineered peptide internal standard preparation:

[0231] 2.5 mL of 5% acetonitrile and 0.1% formic acid were added to the lyophilized TTPP peptide (49 nmol) to achieve a concentration of 19.6 pmol / l L. On the day of use, the concentration of the TTPP peptide was diluted to 0.08 pmol / μL in 40% acetonitrile containing 0.1% formic acid.

[0232] An automated liquid handling platform (Agilent BRAVO TM ) for the quantification of three specific Heterospecific antibodies

[0233] Affinity purification

[0234] For each calibration curve sample or serum sample to be tested, 10 μL of each sample was added to a separate well in a 96-well microplate. Next, 10 μL of additional mouse serum and 30 μL of 0.2 μg / μL stable isotope-labeled universal monoclonal antibody internal standard were added to each well. The 96-well plate was placed on an automated liquid handling platform (Agilent BRAVO TM ) and run the affinity purification protocol consisting of the following steps:

[0235] 1. Initial Syringe Washing

[0236] 2. Perfusion (100 μL PBS at 300 μL / min)

[0237] 3. Equilibration (50 μL PBS at 10 μL / min)

[0238] 4. Load sample (20 μL at 5 μL / min)

[0239] 5. Cup wash 1 (25 μL deionized water)

[0240] 6. Internal box wash 1 (50 μL 1M NaCl in PBS at 10 μL / min)

[0241] 7. Cup wash 2 (25 μL deionized water)

[0242] 8. Internal box wash 2 (50 μL PBS at 10 μL / min)

[0243] 9. Stringent syringe washing (50 μL 5% acetic acid)

[0244] 10. Elution (10 μL 5% acetic acid at 5 μL / min)

[0245] Digestion:

[0246] The 96-well plate containing the eluted samples was placed on an automated liquid handling platform (Agilent BRAVO TM ) on a worktop and perform a digestion protocol comprising the following steps:

[0247] 1. Transfer 40 μL of 8 M urea in 550 mM Tris pH 8 to each well.

[0248] 2. Transfer 4.3 μL of 125 mM DTT to the wells and incubate at 40°C for 60 minutes.

[0249] 3. Transfer 4.7 μL of 250 mM IAM to the wells and incubate at 25°C for 60 minutes.

[0250] 4. Transfer 111 μL of 50 mM ammonium bicarbonate to each well.

[0251] 5. Transfer 20 μL of 0.5 μg / μL trypsin (1:10 enzyme:substrate) and 0.2 μg / μL rLysC (1:25 enzyme:substrate) to each well and incubate at 37°C for 120 minutes.

[0252] 6. Transfer 20 μL of 10% TFA to each well.

[0253] 7. Transfer 30 μL of 0.08 pmol / μL heavily labeled engineered peptide (peptide internal standard) to each well.

[0254] C18 purification:

[0255] Place the plate containing the digested samples on the deck of the Agilent Bravo system and run the "Peptide Cleanup" protocol consisting of the following steps:

[0256] 1. Initial Syringe Washing

[0257] 2. Perfusion (60% acetonitrile with 0.1% TFA)

[0258] 3. Equilibration (50 μL 0.1% TFA at 25 μL / min)

[0259] 4. Load sample (190 μL at 5 μL / min)

[0260] 5. Cup washing (50 μL deionized water)

[0261] 6. Internal cartridge wash (50 μL 0.1% TFA at 25 μL / min)

[0262] 7. Stringent syringe wash (50 μL 60% acetonitrile with 0.1% TFA)

[0263] 8. Elution (20 μL 60% acetonitrile with 0.1% TFA at 5 μL / min)

[0264] LC-MS / MS:

[0265] Each eluted sample (20 μL in 60% acetonitrile containing 0.1% TFA) was immediately diluted to 100 μL with a solution of 35% acetonitrile and 0.1% formic acid to obtain 40% acetonitrile and 0.1% formic acid. Each sample was transferred to a separate LC-MS vial and analyzed on the LC-MS / MS system. Retention time, parent mass, transition and optimized fragmentation conditions vary depending on the peptide sequence and the instrument used for analysis. The TTPP peptide (TTPPVLDSDGSFFLVSK (SEQ ID NO: 7)) was eluted with SCIEX 6500LC-MS / MS system coupled ACQUITY Analyses were performed on an I-Class LC system:

[0266] Column: Waters Acquity UPLC Peptide BEH C18 (2.1 x 150 mm, 1.7 μm)

[0267] Column temperature : 30℃

[0268] Sample injection gradient : Start gradient and hold at 5% B for 1 minute. Increase to 40% B in 17 minutes. Flow rate is 0.25 mL / min. Increase flow rate to 0.4 mL / min. And 90% B for blank injection.

[0269] Blank injection gradient : Start gradient and hold at 90% B for 5 minutes. Flow rate is 0.4 mL / min. Reduce flow rate to 0.25 mL / min. and hold at 5% B for 3 minutes.

[0270] Mobile phase :A is water containing 0.1% formic acid; B is acetonitrile containing 0.1% formic acid

[0271] Retention time : 12.9 minutes

[0272] Table 8. Mass spectrometry settings

[0273] Q1 Quality Q3 Quality Dispersion potential Collision Energy Inlet voltage Collision cell outlet potential Light peptide 905.5 804.4 64 39 10 16 Heavy Peptide 909.5 808.4 64 40 13 16

[0274] For this example, calibration curve samples and 6 serum samples were analyzed on 2 different days. Mice were dosed with trispecific antibodies twice weekly by intraperitoneal injection for a total of 7 doses. Study samples analyzed by LC-MS / MS were collected at sacrifice (approximately 30 hours after the last dose), including control animals dosed with saline (i.e., Fig.21A and 21B 6 mice in the experiment) and 5 mice administered 30 mg / kg of the trispecific antibody (i.e., Fig.21A and 21B 1-5) in mice.

[0275] After LC-MS / MS, each serum calibration curve was analyzed by linear regression analysis with 1 / x weighting by plotting the ratio of the TTPP peptide to its heavy labeled peptide internal standard versus theoretical concentration. Fig. 20A and 20B The results of this analysis are shown. Both serum curves demonstrated good linearity (r-squared ≥ 0.99) and bias (≤ 25%) for the concentration range of 0.012 μg / mL to 800 μg / mL.

[0276] like Fig.21A As shown, serum samples showed higher levels of trispecific peptides in most mice dosed with trispecific antibodies (#1-5) and lower levels in mice dosed with saline (#6). The peptide levels detected in the LC-MS / MS-based assay were consistent between the two assays ( Fig.21A ). These samples were also analyzed using ELISA (n=4 replicates), and the results of the ELISA analysis are shown in Fig.21B The relative levels of the trispecific antibodies detected by LC-MS / MS or ELISA showed a consistent trend for all samples.

[0277] Although the present disclosure includes multiple embodiments, it should be understood that various changes and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent changes that fall within the scope of the present disclosure. In addition, the section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described.

[0278] All references cited in this application are expressly incorporated by reference herein.

Claims

1. A method for quantifying the amount of a polypeptide comprising a portion of an antibody heavy chain constant region in a sample, the method comprising: (a) digesting the sample comprising a polypeptide comprising a portion of an antibody heavy chain constant region, wherein the portion of the antibody heavy chain constant region comprises an engineered mutation, and wherein the digestion produces peptide fragments derived from the antibody heavy chain constant region, the peptide fragments being 5-26 amino acids in length and comprising the engineered mutation, (b) analyzing the digested sample by mass spectrometry to determine the amount of the peptide fragments, thereby determining the amount of the polypeptide comprising a portion of the antibody heavy chain constant region in the sample. 2 . The method according to claim 1 , wherein the peptide fragment does not contain methionine (M) or cysteine ​​(C).

3. The method of claim 1 or claim 2, wherein the peptide fragment does not contain an asparagine (N) followed by a glycine (G) or a serine (S).

4. The method according to any one of claims 1-3, further comprising purifying and concentrating the digested sample prior to the mass spectrometry analysis.

5. The method of claim 4, wherein the digested sample is purified and concentrated by solid phase extraction (SPE).

6. The method according to claim 4, wherein the digested sample is purified and concentrated by SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).

7. The method according to any one of claims 1 to 6, wherein the sample is a whole blood sample, a serum sample, a plasma sample or a tissue sample.

8. The method according to any one of claims 1-7, wherein the sample is from a mouse, a non-human primate or a human.

9. The method of claim 8, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey.

10. The method according to any one of claims 1 to 9, wherein the sample is digested with at least one enzyme.

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