T cell engager masking molecules

Abstract

The present invention relates to a T cell engager masking molecule and a related method for reducing the severity of cytokine release syndrome, wherein the molecule comprises (i) a binding peptide that binds to the T cell engaging paratope of a bispecific T cell engager molecule (TCE), (ii) a linker, and (iii) a half-life extension polymer.

Description

[Technical Field]

[0001] The present invention relates particularly to biotechnologies and medical aspects for preventing cytokine release and / or reducing the severity of cytokine release. [Background technology]

[0002] The emergence of T-cell toxic redirects in the field of immunotherapy has led to a significant increase in the number of treatment approaches over the past year. Beyond genetically modified T cells with chimeric antigen receptors (CARs), various bispecific T-cell engagers (TCEs) have successfully entered the market and are under development to treat a variety of conditions, including cancer. A typical bispecific molecule is a recombinant protein construct formed from binding domains derived from two mobilely linked antibodies. One binding domain of a TCE is typically specific to a selected tumor-associated surface antigen on target cells; the second binding domain is specific to CD3, a subunit of the T-cell receptor (TCR) complex on T cells.

[0003] However, T cell redirection-on-target activity, such as that of CAR T cells, inevitably carries the risk of leading to potent release of pro-inflammatory cytokines that can induce cytokine release syndrome (CRS). CRS is considered a major dose-limiting clinical toxicity associated with T cell-directed immunotherapy that limits the patient's ability to rapidly and safely achieve effective TCE administration (Shimabukuro-Vornhagen, A. et al., (2018) Cytokine release syndrome. Journal for ImmunoTherapy of Cancer 6:56). While the relationship between TCE exposure and CRS is still under investigation, the pathogenesis of CRS may stem from: (i) clinical CRS is primarily associated with the first cycle of TCE therapy; (Shimabukuro-Vornhagen, A., et al., above). (ii) Cytokine release tends to decrease with repeated TCE treatment cycles; (Chen, X. et al., (2019) A Modeling Framework to Characterize Cytokine Release upon T-Cell-Engaging Bispecific Antibody Treatment: Methodology and Opportunities. 12, 600-608); and (iii) TCE regimens including stepwise dosing may reduce CRS incidence, but the success of this dosing strategy may depend on specific targets / symptoms (Jacobs, K. et al., (2017) Lead-in Dose Optimization to Mitigate Cytokine Release Syndrome in AML and MDS Patients Treated with Flotetuzumab, a CD123 x CD3 Dart® Molecule for T-Cell Redirected Therapy. Blood 130, 3856-3856).

[0004] Even if CRS can exist as a transient condition, and even if it is most frequently associated with the first drug or treatment cycle, more severe CRS may require intensive care, lead to interruption of T-cell redirection therapy, and in some cases, can result in patient death if not properly managed. Clinical strategies for managing CRS during immunotherapy are known and include: (a) modifications to the drug regimen, such as longer step-by-step dosing or extension of the intravenous (eIV) administration protocol; (b) prophylactic treatment with corticosteroids (e.g., dexamethasone); and (c) interventional treatment with targeted anti-cytokine antibody drugs (e.g., anti-IL-6R, tocilizumab). Other strategies include target cell pre-depletion approaches and / or the use of target cell masking antibodies, modification of the CD3 binder of T cell engagers to reduce initial infusion cytokine release, or the use of small molecule kinase inhibitors to prevent cytokine release after treatment with T cell engaging therapy (G Leclercy et al. Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors, ONCOIMMUNOLOGY). (See 2022, VOL.11, NO.1). At the molecular design level, masking of TCEs, e.g., AMX-818, ANX007, and other clinical candidates is currently being investigated to mitigate potent on-target and off-tumor toxicity. However, such approaches generally rely on protease-mediated mask cleavage to induce TCE activity. Therefore, one or more protease cleavage sites must be introduced into the linker covalently bound to the masking region and the drug molecule to be masked. These one or more cleavage sites are cleaved by proteases that are frequently dysregulated in tumors, but are significantly more likely to be dysregulated or overexpressed in the tumors actually targeted in each and all cases.In addition, the covalently attached masking moiety requires significant molecular modifications of the specific drug molecule that can affect the efficacy and / or stability properties of the drug molecule without the original masking. Also, for each drug molecule, technical effort must be repeatedly devoted to masking according to the purpose.

[0005] However, despite the progress in CRS management and current technical approaches, known mitigation efforts can reduce or completely eliminate the effectiveness of immunotherapy treatments. Also, the targeted approach is generally not sufficient to prevent the strong and rapid onset of cytokine storms that can occur after CAR T cell treatment or that can be caused by molecules modified by high binding activity to tumor-associated antigens and / or, for example, CD3ε of TCR. Therefore, there is a strong need for improvement in multi-purpose CRS mitigation strategies. There is a need for CRS mitigation strategies applicable to various TCIs, such as the transient inhibition of a wide range of cytokines and chemokines to more efficiently dissipate CRS symptoms, as observed with glucocorticoids.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0007] Surprisingly, TCE masking molecules that are combined with TCE, for example when co-administered with TCE, are functionalized with polymers to extend their half-life, and generally contain binding peptides derived from CD3 T cell coreceptors, e.g., the endogenous N-terminal sequence of the CD3ε T cell coreceptor, are effective against maximum TCE exposure (C max The gradual start of the attenuation of (t max This can effectively suppress the pharmacokinetic (PK) exposure profile of TCEs, which is characterized by [specific features].

[0008] Therefore, in one aspect, this disclosure is, A T cell engager (TCE) masking molecule, (i.) At least one binding peptide that binds to the T cell engaging paratope of a T cell engager (TCE) molecule (this T cell engaging paratope is an anti-CD3 paratope to an epitope located within CD3δ (SEQ ID NO: 256), CD3ε (SEQ ID NO: 257), (ii) at least one linker covalently bonded to the C-terminus of the peptide; (iii) At least one half-life-extending polymer, preferably with a half-life of at least 2 kDa, which is covalently bonded to the linker. This provides a T cell engager (TCE) masking molecule that includes [specific component].

[0009] According to the above embodiment, it is also conceivable that the binding peptide may bind to the anti-CD3 paratope of TCE (the anti-CD3 paratope binds to an epitope containing at least one residue selected from CD3ε (SEQ ID NO: 257): K73 and S83; and CD3δ (SEQ ID NO: 256): K82 and C93, which preferably includes the CD3ε region defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82 and S83, and which includes the CD3δ region defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92 and C93).

[0010] In another aspect of this disclosure, the TCE masking molecule is (i) a binding peptide that binds to the anti-CD3 epsilon (CD3ε) paratope of the CD3ε-binding domain of TCE (this anti-CD3ε paratope is against an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257); (ii) A linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life-extending polymer covalently bonded to the linker, Includes.

[0011] According to the above embodiment, it is also conceivable that the anti-CD3ε paratope is an amino acid sequence consisting of SEQ ID NO: 258 or a shorter N-terminal sequence of SEQ ID NO: 258, preferably a CD3ε epitope consisting of the amino acid sequence of SEQ ID NO: 259 or QDGNEE, QDGNEEM, or QDGNEEMG.

[0012] According to the above embodiment, the bound peptide may also contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids, preferably 5, 6, 7, 8, 9, or 10 amino acids, more preferably 10 amino acids.

[0013] According to the above embodiment, the bound peptide is bound to the anti-CD3ε paratope and contains at least the sequence X1DGX2E (SEQ ID NO: 260) at its N-terminus, where X1 is selected from Q, pyroglutamic acid (pE), and S, and X2 is also expected to be selected from N, E, S, T, V, and I.

[0014] According to the above embodiment, the binding peptide is also envisioned to bind to the anti-CD3ε paratope and to contain at least the sequence X1X2X3X4EX5 (SEQ ID NO: 392) at its N-terminus (where X1 is Q, pyroglutamic acid (pE), or S; X2 is D, H, or N; X3 is G, F, or Y; X4 is N, E, S, T, V, or I; and X5 is E, L, P, or W).

[0015] According to the above embodiment, the binding peptide is also envisioned to bind to the anti-CD3ε paratope and to contain at least the sequence X1DGX2EE (SEQ ID NO: 261) (where X1 is Q, pE, or S, and X2 is N, E, S, T, V, or I) at its N-terminus.

[0016] According to the above embodiment, the binding peptide is also envisioned to bind to the anti-CD3ε paratope and to contain at least the sequence X1X2X3X4EX5X6X7 (SEQ ID NO: 393) at its N-terminus (where X1 is Q, pyroglutamic acid (pE), or S; X2 is D, H, or N; X3 is G, F, or Y; X4 is N, E, S, T, V, or I; X5 is E, L, P, or W; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably pEX2X3X4EEX6X7, and more preferably pEDGX4EEX6X7).

[0017] According to the above embodiment, the bound peptide is also assumed to be bound to the anti-CD3ε paratope and to contain at least the sequence pEX1X2X3EX4LK (SEQ ID NO: 396) at its N-terminus (where X1 is D, H, or N; X2 is G, F, or Y; X3 is N, E, S, T, V, or I; and X4 is E, L, P, or W).

[0018] According to the above embodiment, it is also conceivable that the anti-CD3ε binding peptide may include a palindrome.

[0019] According to the above embodiment, it is also conceivable that the palindrome has K at a position between the mirror-image amino acids.

[0020] According to the above embodiment, the binding peptide is bound to the anti-CD3ε paratope and may contain at least one of sequence numbers 263-284, 286-293, 295-303, 308-338, 385-388, 397-419, and 431, or any of sequence numbers 263-284, 286-293, 295-338, 385-388, 397-419, and 431 at its N-terminus.

[0021] According to the above embodiment, the C-terminal bound peptide may further contain amino acids G and C or G and K.

[0022] According to the above embodiment, the bound peptide is bound to the anti-CD3ε paratope and is pEX1X2X3EX4X5X6GX7 (SEQ ID NO: 433) (where X1 is D, H or N, X2 is G, F or Y, preferably G or F, X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y or I, preferably N, E, H, Q or R, and X4 is E, L, P or It is also conceivable that the sequence may include, preferably pEX1X2X3EEX5X6GX7, more preferably pEDGX3EEX5X6GX7 (where W is and X5 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, and X7 is C or K).

[0023] According to the above embodiment, the bound peptide may also be bound to the anti-CD3ε paratope and contain the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (wherein X1 is D, H, or N; X2 is G, F, or Y, preferably G or F; X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E; and X4 is C or K).

[0024] According to the above embodiment, the binding peptide may also be bound to the anti-CD3ε paratope and contain the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (wherein X1 is D, H, or N; X2 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably E, N, H, Q, or R, most preferably N or E; and X3 is C or K).

[0025] According to the above embodiment, the binding peptide may also be bound to the anti-CD3ε paratope and contain the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (wherein X1 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E, and X2 is C or K).

[0026] According to the above embodiment, the binding peptide is also expected to bind to the anti-CD3ε paratope and include the sequence of SEQ ID NOs: 297, 300, 303, 304, 305, 306, 307, 384, or 389.

[0027] According to the above embodiment, the linker may also be selected from the group consisting of (i) a disulfide linker containing a disulfide (RSS-R') (where R is a half-life extension polymer and R' is a binding peptide); (ii) a free thiol containing linear or branched PEG, OPSS derivatives, maleimide, norbornene, acrylimide, acrylate, vinyl sulfone, and amine; and (iii) a carboxyl linker containing amines, amides, and epsilon-derivativeized acetyl bromide.

[0028] According to the above embodiment, the linker comprises the following parts: [ka] It is also conceivable that the following could be selected (wherein R is a half-life extension polymer and R' is a CD3ε masking peptide).

[0029] According to the above embodiment, the linker is formed when the C-terminal amino acid of the binding peptide is K, by formula [ka] A maleimide-thiosuccinimide linker having a binding peptide, where the C-terminal amino acid of the binding peptide is C, is given by the formula [ka] It is also conceivable that a disulfide having the formula R'-SSR may be selected from acetamie-thioethers having a methyl

[0030] According to the above embodiment, the half-life-extending polymer may also be selected from mono-methoxypolyethylene glycol (mPEG), linear, 2-arm, 4-arm, and 8-arm polyethylene glycol (PEG); PLGA; peptide acrylate, polyglycerol, polyoxazoline, polyvinylpyrrolidone, polyacrylamide, poly(N-acryloylmorpholine), poly(N,N-dimethylacrylamide), ply(ply)(2-hydroxypropyl methacrylamide), polysarcosine, poly(2-hydroxyethyl methacrylamide), hyaluronic acid, sialic acid, poly[(organo)phosphazene], and heparin.

[0031] According to the above embodiment, the half-life extension polymer comprises the following parts: [ka] (wherein n is an integer between approximately 20 and approximately 200, preferably between 20 and 169, more preferably between approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 103, 110, or 113) [ka] (wherein n is an integer between approximately 40 and approximately 400, preferably between approximately 40 and approximately 208 or between approximately 57 and approximately 338, more preferably between approximately 50, 57, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or approximately 225) and (iii.)(CH2CH2O)n (wherein n is an integer between approximately 80 and approximately 700, preferably between approximately 80 and approximately 675, more preferably approximately 80, 83, 90, 100, 110, 113, 120, 130, 140, 150, 160, 166, 170, 180, 190, 200, 400, 410, 417, 420, 430, 440, 450, 460, or approximately 470) It is also possible that the PEG is selected from among these options.

[0032] According to the above embodiment, the half-life extension polymer may be a non-branched linear or branched 2-arm, 4-arm or 8-arm PEG, preferably linear or 4-arm, with a molecular weight of about 2 kDa to about 60 kDa, preferably about 4 kDa to about 30 kDa or 10 kDa to about 30 kDa, or more preferably about 5 or about 20 kDa.

[0033] According to the above embodiment, the half-life extension polymer may also be a branched polymer, preferably a 2-arm, 4-arm, and / or 8-arm PEG, in which a plurality of binding peptides are linked via linkers, preferably 2, 4, or 8 binding peptides are linked to one half-life extension polymer, and having a molecular weight of about 2 to 60 kDa.

[0034] According to the above embodiment, the bound peptide contains the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (wherein X1 is D, H or N, X2 is G, F or Y, preferably G or F, and X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y or I, preferably N, E, H, Q or R, most preferably N or E), (a.) When X4 is C; formula [ka] A linker selected from maleimide-thiosuccinimide linkers having the formula R'-SSR and disulfides having the formula R'-SSR; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] (b) a half-life extension polymer which is PEG, and (b) if X4 is K; formula [ka] An acetamide-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] It is also conceivable that it may include a half-life extension polymer that is a PEG.

[0035] According to the above embodiment, the bound peptide contains the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (wherein X1 is D, H, or N, and X2 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably E, N, H, Q, or R, most preferably N or E), (a.) Here, if X3 is C; formula [ka] Maleimide-thiosuccinimide linker and a linker selected from disulfides having the formula R'-SSR; and the following formula having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. [ka] (b) a half-life extension polymer which is PEG, and (b) if X3 is K; formula [ka] An acetamide-thioether having; and a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] The PEG is a half-life extended polymer and contains (where R' represents the bound peptide and R represents the half-life extended PEG polymer) This is also a possibility.

[0036] According to the above embodiment, the sequence pEDGX1EELKGX2 (sequence number 436) (where X1 is N or E, and (a) X2 is C) and the formula [ka] A linker selected from maleimide-thiosuccinimide linkers having the formula R'-SSR and disulfides having the formula R'-SSR; and a linker having a molecular weight of approximately 5 kDa or approximately 20 kDa, the following formula [ka] (b) a half-life extension polymer which is PEG, comprising; or the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is N or E and X2 is K); formula [ka] An acetamide-thioether having; and a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] It is also conceivable that the product may contain a half-life extension polymer that is a PEG (where R' represents the binding peptide and R represents the half-life extension PEG polymer).

[0037] According to the above embodiment, the masking molecule is (i.) A conjugated peptide that binds to the anti-CD3ε paratope and contains any one amino acid sequence of SEQ ID NOs. 263-284, 286-293, 295-303, 304-307, 308-338, 385-388, 397-419 and 431; (ii.) A linker covalently bonded to the C-terminus of the binding peptide; (iii) At least one half-life-extending polymer, preferably with a half-life of at least 2 kDa, covalently bonded to the linker, It is also hypothesized that it may contain T cell engager masking molecules.

[0038] According to the above embodiment, the masking molecule (a.) The amino acid sequence of sequence numbers 304, 306, 384, or 389, formula [ka] A linker selected from a maleimide-thiosuccinimide linker having the formula R'-SSR and a disulfide having the formula R'-SSR; The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula [ka] A PEG half-life extension polymer, or, (b) The amino acid sequence of SEQ ID NO: 305 or 307; formula [ka] An acetamide-thioether having, The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula [ka] It is also conceivable that the T cell engager masking molecule may include a half-life extension polymer that is PEG, and (where R' represents the binding peptide and R represents the half-life extension PEG polymer).

[0039] According to the above embodiment, the masking molecule (i.) A conjugated peptide having sequence number 304; (ii.) The formula is covalently bonded to the C-terminus of the binding peptide. [ka] Aleimide-thiosuccinimide linker Or disulfide R'-SSR and; (iii.) The following formula has a molecular weight of 5 kDa or 20 Daa. [ka] Half-life extension polymers and It is also hypothesized that it may contain T cell engager masking molecules.

[0040] According to the above embodiment, the masking molecule (i.) A conjugated peptide having sequence number 305; (ii.) The formula is covalently bonded to the C-terminus of the binding peptide. [ka] acetamide-thioether having and; (iii.) The following formula has a molecular weight of 5 kDa or 20 Daa. [ka] [ka] Half-life extension polymers and It is also hypothesized that it may contain T cell engager masking molecules.

[0041] According to the above embodiment, the masking molecule (i.) A conjugated peptide having sequence number 306; (ii.) Formula [ka] A maleimide-thiosuccinimide linker having or (or) a disulfide R'-SSR covalently bonded to the C-terminus of the binding peptide ; (iii.) The following formula has a molecular weight of 5 kDa or 20 Daa. [ka] [ka] Half-life extension polymers and It is also hypothesized that it may contain T cell engager masking molecules.

[0042] According to the above embodiment, the masking molecule (i.) A conjugated peptide having sequence number 307; (ii.) The formula is covalently bonded to the C-terminus of the binding peptide. [ka] acetamide-thioether having ; (iii.) The following formula has a molecular weight of 5 kDa or 20 Daa. [ka] [ka] Half-life extension polymers and It is also hypothesized that it may contain T cell engager masking molecules.

[0043] According to the above embodiment, in Table 1, the TCE masking molecule may also include any of the combinations (a.) to (k.) of the binding peptide, linker, and half-life extending polymer (where R' represents the binding peptide and R represents the half-life extending polymer):

[0044] [Table 1]

[0045] [Table 2]

[0046] According to the above embodiment, it is also conceivable that the TCE masking molecule includes any of the combinations (a.) to (k.) of a binding peptide, a linker, and a half-life-extending PEG polymer (where R' represents the binding peptide and R represents the half-life-extending PEG polymer).

[0047] [Table 3]

[0048] [Table 4]

[0049] [Table 5]

[0050] According to the above embodiment, it is also conceivable that the half-life of the TCE masking molecule is significantly shorter than that of TCE, which is about 1 to about 48 hours, preferably about 1.5 to about 24 hours, more preferably about 1.5 to about 4 hours, or about 2 hours.

[0051] According to the above embodiment, the binding peptide binds to the anti-CD3ε paratope of the CD3ε binding domain of TCE with affinity for Kd values ​​typically less than 10 nM, less than 5 nM, less than 0.5 nM, or about 0.1 nM to 5 nM. Preferably, this can be achieved when the amino acid sequence of the binding peptide is, for example, pEX1GX2EELKGX3 (SEQ ID NO: 435) (where X1 is D, H, or N, and X2 is N).

[0052] According to the above embodiment, when the amino acid sequence of the binding peptide is pEX1GX2EELKGX3 (SEQ ID NO: 435) (where X1 is D, H, or N, and X2 is E), the binding peptide typically binds to the anti-CD3ε paratope of the CD3ε binding domain of TCE with an affinity of approximately 10 to 100 nM.

[0053] Another aspect of the present invention also envisions providing a method for reducing the severity of cytokine release or cytokine release syndrome in human subjects treated with TCE, the method comprising administering an effective dose of a T cell engager masking molecule to a human subject, the TCE masking molecule being (i.) A binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of TCE (the anti-CD3ε paratope is against an epitope located within CD3ε, where CD3ε contains the amino acid sequence of SEQ ID NO: 257); (ii.) A linker covalently bonded to the C-terminus of the binding peptide; (iii) At least one half-life-extending polymer, preferably with a half-life of at least 2 kDa, which is covalently bonded to the linker. Includes.

[0054] According to the above embodiment, the T cell engager masking molecule is The compound peptide contains a conjugated peptide that binds to the anti-CD3ε paratope and comprises the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (where X1 is D, H, or N; X2 is G, F, or Y, preferably G or F; and X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R), (a.) when X4 is C; formula [ka] Maleimide-thiosuccinimide linker and a linker selected from disulfides having the formula R'-SSR; and the following formula having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. [ka] (b) a half-life extension polymer which is PEG, and (b) if X4 is K; formula [ka] An acetamide-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] It contains a half-life extended polymer that is PEG (where R' represents the bound peptide and R represents the half-life extended PEG polymer).

[0055] According to the above embodiment, the T cell engager masking molecule is The anti-CD3ε paratope contains a conjugated polypeptide comprising the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (wherein X1 is D, H, or N, and X2 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, H, Q, or R), and (a.) when X3 is C, the formula [ka] A linker selected from maleimide-thiosuccinimide linkers having the formula R'-SSR and disulfides having the formula R'-SSR; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] (b) a half-life extension polymer which is PEG, and (b) if X3 is K; formula [ka] An acetamide-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] It contains a half-life extended polymer that is PEG (where R' represents the bound peptide and R represents the half-life extended PEG polymer).

[0056] According to the above embodiment, the T cell engager masking molecule is: The anti-CD3ε paratope contains a conjugated peptide comprising the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is D, H, or N, and X1 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E), and (a.) when X2 is C; formula [ka] A linker selected from maleimide-thiosuccinimide linkers having the formula R'-SSR and disulfides having the formula R'-SSR; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] (b) a half-life extension polymer which is PEG, and (b) if X2 is K; formula [ka] An acetamide-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [ka] It contains a half-life extended polymer that is PEG (where R' represents the bound peptide and R represents the half-life extended PEG polymer).

[0057] According to the aforementioned aspect, the T cell engager masking molecule is (a) The amino acid sequence of SEQ ID NOs. 304, 306, 384, or 389, formula [ka] A linker selected from a maleimide-thiosuccinimide linker having the formula R'-SSR and a disulfide having the formula R'-SSR; The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula [ka] A half-life extension polymer that is PEG, or (b) The amino acid sequence of SEQ ID NO: 305 or 307; formula [ka] An acetamide-thioether having, The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula [ka] It contains a half-life extended polymer that is PEG (where R' represents the bound peptide and R represents the half-life extended PEG polymer).

[0058] According to the above embodiment, a method for reducing the severity of cytokine release in human subjects, preferably those being treated with TCE, may include the use or administration of a TCE masking molecule comprising any of the combinations (a.) to (k.) of a binding peptide, a linker, and a half-life extending polymer (where R' represents the binding peptide and R represents the half-life extending polymer).

[0059] [Table 6]

[0060] [Table 7]

[0061] [Table 8]

[0062] According to the above embodiment, a method for reducing the severity of cytokine release in a human subject, preferably one being treated with TCE, includes the use or administration of any combination (a.) to (k.) of a binding peptide, a linker, and a half-life extended PEG polymer (where R' represents the binding peptide and R represents the half-life extended PEG polymer).

[0063] [Table 9]

[0064] [Table 10]

[0065] According to the above embodiment, the immunotherapy includes administering a T-cell engager.

[0066] According to the above embodiment, the TCE masking molecule is administered before, during, or after the administration of TCE.

[0067] According to the above embodiment, the molar ratio of the masking molecule to TCE is also assumed to be in the range of 1000:1 to 10:1 or 250:1 to 10:1, preferably 100:1 to 25:1.

[0068] According to the above embodiment, TCE is (ii) comprising at least three domains in order from amino to carboxyl, (a) The first domain binds to a target cell surface antigen, which is preferably a tumor antigen; (b) The second domain binds to an extracellular epitope of the human and / or macaque CD3 chain, preferably CD3ε; (c.) The third domain comprises two polypeptide monomers, each containing a hinge, a CH2 domain, and a CH3 domain, the two polypeptide monomers fused to each other via a peptide linker, and the third domain is sequentially from amino to carboxyl: Includes hinge-CH2-CH3-linker-hinge-CH2-CH3, TCE; (ii.) (a.) A first binding domain that binds to a first target cell surface antigen (e.g., TAA1), (b.) A second binding domain that binds to the extracellular epitope of CD3ε, (c) Spacers that are single-chain fragment crystallizable regions (scFc), human serum albumin (HSA), programmed death receptor 1 (PD1), or heterofragment crystallizable regions (heteroFC); (d.) A third binding domain that binds to a second target cell surface antigen (e.g., TAA2), and (e.) A fourth binding domain that binds to the extracellular epitope of CD3ε. TCE including, The first binding domain binds to the first target cell surface antigen, and the third binding domain simultaneously binds to the second target cell surface antigen, where the first and second target cell surface antigens are on the same target cell. TCE is a single polypeptide chain, and the first target cell surface antigen and the second target cell surface antigen are not identical. The first binding domain and the second binding domain form the first dual specificity entity, the third binding domain and the fourth binding domain form the second dual specificity entity, and The spacer entity is located between the first bispecific portion and the second bispecific portion. TCE; and (iii) Full-length bispecific antibody based on IgG; or (iv.) Heterodimers It is also possible that the selection will be made from these options.

[0069] According to the above embodiment, it is also conceivable that the TCE under (i.) or (ii.) is a single-chain molecule.

[0070] According to the above embodiment, the glycosylation site at the Kabat position 314 of the CH2 domain in the third domain of the bispecific antigen-binding molecule is removed by N314X substitution, where X is any amino acid except Q.

[0071] According to the above embodiment, each of the polypeptide monomers of the third domain may have amino acids that are at least about 80, 85, 90, 95, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 437 to 444, or it may have a sequence amino acid sequence selected from the group consisting of SEQ ID NOs: 437 to 444.

[0072] According to the above embodiment, the CH2 domain may also include intradomain cysteine ​​disulfide bridges.

[0073] According to the above embodiment, it is also conceivable that the tumor antigen may be selected from the group consisting of CDH19, CDH3, MSLN, DLL3, FLT3, EGFRvIII, BCMA, PSMA, CD33, CD19, CD20, CLDN18.2, CLDN6, MUC17, EpCAM, STEAP1, and CD70.

[0074] According to the above embodiment, the antibody construct is arranged sequentially from amino to carboxyl: (a) The first domain; (b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 187-189; (c) Second domain; (d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) A first polypeptide monomer of a third domain having any of the amino acid sequences of SEQ ID NOs. 437-444; (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) A second polypeptide monomer of the third domain having one of the amino acid sequences of SEQ ID NOs. 437-444. It is also possible that this may be included.

[0075] According to the above embodiment, the first binding domain of the construct may also include a VH region containing CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of the following, and a VL region containing CDR-L1, CDR-L2, and CDR-L3: (a) CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, CDR-H3 as shown in SEQ ID NO: 6, CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3 (b) CDR-H1 as shown in SEQ ID NO: 29, CDR-H2 as shown in SEQ ID NO: 30, CDR-H3 as shown in SEQ ID NO: 31, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 as shown in SEQ ID NO: 35, and CDR-L3 as shown in SEQ ID NO: 36 (c) CDR-H1 as shown in sequence number 42, CDR-H2 as shown in sequence number 43, CDR-H3 as shown in sequence number 44, CDR-L1 as shown in sequence number 45, CDR-L2 as shown in sequence number 46, and CDR-L3 as shown in sequence number 47 (d) CDR-H1 as shown in sequence number 53, CDR-H2 as shown in sequence number 54, CDR-H3 as shown in sequence number 55, CDR-L1 as shown in sequence number 56, CDR-L2 as shown in sequence number 57, and CDR-L3 as shown in sequence number 58, (e) CDR-H1 as indicated by sequence number 65, CDR-H2 as indicated by sequence number 66, CDR-H3 as indicated by sequence number 67, CDR-L1 as indicated by sequence number 68, CDR-L2 as indicated by sequence number 69, and CDR-L3 as indicated by sequence number 70, (f) CDR-H1 as shown in sequence number 83, CDR-H2 as shown in sequence number 84, CDR-H3 as shown in sequence number 85, CDR-L1 as shown in sequence number 86, CDR-L2 as shown in sequence number 87, and CDR-L3 as shown in sequence number 88, (g) CDR-H1 as indicated by SEQ ID NO: 94, CDR-H2 as indicated by SEQ ID NO: 95, CDR-H3 as indicated by SEQ ID NO: 96, CDR-L1 as indicated by SEQ ID NO: 97, CDR-L2 as indicated by SEQ ID NO: 98, and CDR-L3 as indicated by SEQ ID NO: 99 (h) CDR-H1 as indicated by sequence number 105, CDR-H2 as indicated by sequence number 106, CDR-H3 as indicated by sequence number 107, CDR-L1 as indicated by sequence number 109, CDR-L2 as indicated by sequence number 110, and CDR-L3 as indicated by sequence number 111, (i) CDR-H1 as indicated by sequence number 115, CDR-H2 as indicated by sequence number 116, CDR-H3 as indicated by sequence number 117, CDR-L1 as indicated by sequence number 118, CDR-L2 as indicated by sequence number 119, and CDR-L3 as indicated by sequence number 120, (j) CDR-H1 as indicated by sequence number 126, CDR-H2 as indicated by sequence number 127, CDR-H3 as indicated by sequence number 128, CDR-L1 as indicated by sequence number 129, CDR-L2 as indicated by sequence number 130, and CDR-L3 as indicated by sequence number 131. (k) CDR-H1 as indicated by sequence number 137, CDR-H2 as indicated by sequence number 138, CDR-H3 as indicated by sequence number 139, CDR-L1 as indicated by sequence number 140, CDR-L2 as indicated by sequence number 141, and CDR-L3 as indicated by sequence number 142, (l) CDR-H1 as shown in sequence number 152, CDR-H2 as shown in sequence number 153, CDR-H3 as shown in sequence number 154, CDR-L1 as shown in sequence number 155, CDR-L2 as shown in sequence number 156, and CDR-L3 as shown in sequence number 157, (m) CDR-H1 as indicated by sequence number 167, CDR-H2 as indicated by sequence number 168, CDR-H3 as indicated by sequence number 169, CDR-L1 as indicated by sequence number 170, CDR-L2 as indicated by sequence number 171, and CDR-L3 as indicated by sequence number 172, (n) CDR-H1 as shown in sequence number 203, CDR-H2 as shown in sequence number 204, CDR-H3 as shown in sequence number 205, CDR-L1 as shown in sequence number 206, CDR-L2 as shown in sequence number 207, and CDR-L3 as shown in sequence number 208, (o) CDR-H1 as indicated by sequence number 214, CDR-H2 as indicated by sequence number 215, CDR-H3 as indicated by sequence number 216, CDR-L1 as indicated by sequence number 217, CDR-L2 as indicated by sequence number 218, and CDR-L3 as indicated by sequence number 219, (p) CDR-H1 as shown in sequence number 226, CDR-H2 as shown in sequence number 227, CDR-H3 as shown in sequence number 228, CDR-L1 as shown in sequence number 229, CDR-L2 as shown in sequence number 230, and CDR-L3 as shown in sequence number 231; (q) CDR-H1 as shown in sequence number 238, CDR-H2 as shown in sequence number 239, CDR-H3 as shown in sequence number 240, CDR-L1 as shown in sequence number 241, CDR-L2 as shown in sequence number 242, and CDR-L3 as shown in sequence number 243, (r) CDR-H1 as indicated by sequence number 420, CDR-H2 as indicated by sequence number 421, CDR-H3 as indicated by sequence number 422, CDR-L1 as indicated by sequence number 423, CDR-L2 as indicated by sequence number 424, and CDR-L3 as indicated by sequence number 425.

[0076] According to the above embodiment, it is also conceivable that the bispecific antigen-binding molecule may have SEQ ID NOs: 17, 52, 63, 81, 93, 104, 114, 125, 136, 147, 162, 177, 213, 226, 238, 248, 255, or 430, preferably 104 or 255.

[0077] According to the above embodiment, the bispecific antigen-binding molecule is a) The first monomer, 1) The first variable heavy chain domain; 2) A first constant heavy chain containing the first CH1 domain and the first Fc domain ; 3) Binds to human CD3, scFv variable light chain, scFv includes an scFv linker and an scFv variable heavy chain domain (the scFv is between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain) (Covalently linked using a domain linker) A first monomer containing a first heavy chain; b) A second monomer comprising a second variable heavy chain domain, a second steady heavy chain comprising a second Fc domain, and a second heavy chain comprising a second heavy chain; c) A common light chain including variable light chain domains and constant light chain domains, It is also assumed that it is a heterodimer antibody containing, Here, the first variable heavy chain domain and the variable light chain domain bind to human STEAP1, and the second variable chain domain and the variable light chain domain bind to human Combine with STEAP1, and here, a) The first monomer contains the sequence of sequence number 375, and the second The monomer contains the sequence of sequence number 374, and the common light chain is Does it contain the sequence with sequence number 373? b) The first monomer contains the sequence of sequence number 380; the second The monomer contains the sequence of sequence number 376; the common light chain is Contains the sequence of sequence number 373; or c) The first monomer contains the sequence of sequence number 379; the second The monomer contains the sequence of sequence number 378, and the common light chain is Contains the sequence with sequence number 377.

[0078] According to the above embodiment, the CD3ε-binding domain of TCE has the sequence of sequence number 26, 381, 382, ​​or 383.

[0079] In another embodiment, a TCE masking molecule is provided for use in reducing the severity of cytokine release in human subjects undergoing TCE treatment, wherein the TCE masking molecule is (i.) A binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of TCE (this anti-CD3ε paratope is against an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257); (ii) A linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life-extending polymer covalently bonded to the linker, Includes.

[0080] In another embodiment, a method for attenuating in vivo exposure to TCE, wherein administration is preferably via the IV or SC pathway, and this method is (a) (i.) A binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of TCE (where the anti-CD3ε paratope is for an epitope located within CD3ε, and CD3ε includes the amino acid sequence of SEQ ID NO: 257), (ii) A linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life-extending polymer covalently bonded to a linker, wherein the binding peptide binds to the anti-CD3ε paratope at a Kd at least 1.5 times lower, preferably at least 2 times lower, more preferably 3 times lower, than the Kd of the amino acid sequence of SEQ ID NO: 262 that binds to the anti-CD3ε paratope. (b) Administer a TCE masking molecule in a molar excess of approximately 10 to 1000 or approximately 25 to 250 with respect to TCE. The procedure includes the following steps. According to the above embodiment, the TCE masking molecule is administered before, during, or after administration of TCE.

[0081] In another embodiment, a method is envisioned for reducing the severity of cytokine release in human subjects undergoing immunotherapy, the method comprising administering an effective dose of a T cell engager masking molecule to the human subject before, concurrently with, or after immunotherapy, wherein the T cell engager masking molecule is (i.) A conjugated peptide having a sequence number selected from the group consisting of sequence numbers 263-284, 286-293, 295-338, 385-388, 397-419 and 431; (ii.) A linker covalently bonded to the C-terminus of the binding peptide; (iii) at least one half-life extension polymer, preferably with a half-life of at least 2 kDa, which is covalently bonded to the linker. Includes.

[0082] In another embodiment, a method is envisioned that involves administering an effective dose of a TCE masking molecule or a pharmaceutically acceptable salt thereof, as disclosed in the specification, to a subject requiring such treatment, preferably in combination with a therapeutically effective dose of a T cell engager in a molar ratio in the range of 1000:1 to 10:1 or 250:1 to 10:1 (masking molecule: TCE). The TCE masking molecule is administered before, during, or after administration of TCE.

[0083] In another embodiment, the TCE masking molecule is intended for use in attenuating in vivo exposure to TCE after administration of the TCE molecule, and this administration is preferably via the IV or SC pathway, and this method is (a) (i.) A binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of TCE (where the anti-CD3ε paratope is against an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257); (ii) A linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life-extending polymer covalently bonded to the linker, A TCE masking molecule containing, The aforementioned binding peptide provides a TCE masking molecule that binds to the anti-CD3ε paratope at a Kd at least 1.5 times lower, preferably at least 2 times lower, and more preferably 3 times lower, than the Kd of the amino acid sequence of SEQ ID NO: 262 that binds to the anti-CD3ε paratope. (b) Administer the TCE masking molecule in a molar excess of approximately 10 to 1000 or approximately 25 to 250 relative to the TCE before, during, or after administration of the TCE. Includes stages. [Brief explanation of the drawing]

[0084] [Figure 1] Figure 1 shows the general pharmacokinetic and pharmacodynamic relationships between TCE exposure and cytokine release. (Left) Relationship between the overall PK profile and cytokine release for TCE administered by an IV bolus. This figure shows TCE administered to maintain a minimum plasma concentration above a predetermined exposure target (EC90). This scenario often leads to patient overexposure, causing a rapid cytokine release early after administration, which can induce runaway CRS. (Right) An alternative scenario in which active TCE is administered more gradually to reduce cytokine stimulation and lower maximum exposure (Cmax), while maintaining TCE coverage above the desired effective concentration to ensure efficacy. [Figure 2] Figure 2 shows a representative in silico PK model that highlights the activity or sensitivity to exposure to "free" TCE when co-administered with the masking molecule CD3ε PepPOL. (Left) Schematic diagram of an in silico PK model defining the relationship between plasma (Cp) and tissue (Cp) "free" TCE concentrations, unbound CD3ε PepPOL (T1), and binding kinetics that promote TCE-(CD3ε PepPOL) complex formation. Note: It is assumed that the elimination rates (kel,D) of TCE and TCE-(CD3ε PepPOL) are identical. It is assumed that the elimination rate of CD3ε PepPOL, kel,T is >>kel,D. (Right) Simulation of TCE administered by IV bolus (10 ug / kg) alone (black) or in combination with CD3ε PepPOL: equimolar dosing, KD=1 nM (orange), equimolar dosing, KD=0.1 nM (blue, solid line); or 1000-fold molar excess, KD=0.1 nM (blue, dotted line) (non-human primate PK parameters for CD20 HLE-BiTE). Note the effects of both affinity and overproduction on both Cmax and tmax from day 1 to day 2 of administration, with very slight exposure disturbances thereafter. [Figure 3] Figure 3 shows the transient association between a representative masking molecule, CD3ε-PepPOL, and a representative TCE molecule with an extended half-life scFc moiety. [Figure 4] Figure 4 shows the representative plasma concentrations over time in C57BL / 6 mice of a masking molecule containing a half-life extension polymer (SEQ ID NO: 297) compared to the binding peptide alone. [Figure 5] Figure 5 shows the effect of representative polymers on masking molecules in mice. [Figure 6] Figure 6 shows the effect of the maleamide linker's PK on representative masking molecules. [Figure 7A] Figure 7 shows the cell-lysing effect of TCE on B cells. [Figure 7B] Figure 7 shows the cell-lysing effect of TCE on B cells. [Figure 8A]Figure 8 shows the reduction in cytokine release upon co-administration of CD3ε PepPol in >1000X peptide:TCE overproduction. [Figure 8B] Figure 8 shows the reduction in cytokine release upon co-administration of CD3ε PepPol in >1000X peptide:TCE overproduction. [Figure 8C] Figure 8 shows the reduction in cytokine release upon co-administration of CD3ε PepPol in >1000X peptide:TCE overproduction. [Figure 8D] Figure 8 shows the reduction in cytokine release upon co-administration of CD3ε PepPol in >1000X peptide:TCE overproduction. [Figure 9A] Figure 9 shows the co-administration of muCD19 TCE + CD3ε-PepPOL, and the co-administration of v1 reduces cytokine release as a function of the CD3ε-PepPOL v1 dose. [Figure 9B] Figure 9 shows the co-administration of muCD19 TCE + CD3ε-PepPOL, and the co-administration of v1 reduces cytokine release as a function of the CD3ε-PepPOL v1 dose. [Figure 9C] Figure 9 shows the co-administration of muCD19 TCE + CD3ε-PepPOL, and the co-administration of v1 reduces cytokine release as a function of the CD3ε-PepPOL v1 dose. [Figure 9D] Figure 9 shows the co-administration of muCD19 TCE + CD3ε-PepPOL, and co-administration of v1 reduces cytokine release as a function of CD3ε-PepPOL and v1 dose. [Figure 9E] Figure 9 shows the co-administration of muCD19 TCE + CD3ε-PepPOL, and co-administration of v1 reduces cytokine release as a function of CD3ε-PepPOL and v1 dose. [Figure 10A] Figure 10 shows the co-administration of muCD19 TCE + CD3ε-PepPol, where v1 reduced cytokine release and had minimal effect on the PD response in the huCD3ε KI model. [Figure 10B]Figure 10 shows the co-administration of muCD19 TCE + CD3ε-PepPol, where v1 reduced cytokine release and had minimal effect on the PD response in the huCD3ε KI model. [Figure 11A] Figure 11 shows the co-administration of CD3ε-PepPOL, where v1 reduced cytokine release while maintaining antitumor activity in a preclinical MC38-muCD19 tumor model. (A) Serum cytokine INF-y; (B) Serum cytokine-releasing TNF-a; (C) Target cell depletion in the spleen and (D) in the blood; (E) Inhibition of tumor growth. Note: Statistical analysis was performed using one-way ANOVA for panels (A) and (B), and repeated-measures two-way ANOVA for panel (C) using GraphPad Prism v 9.5.1. [Figure 11B] Figure 11 shows the co-administration of CD3ε-PepPOL, where v1 reduced cytokine release while maintaining antitumor activity in a preclinical MC38-muCD19 tumor model. (A) Serum cytokine INF-y; (B) Serum cytokine-releasing TNF-a; (C) Target cell depletion in the spleen and (D) in the blood; (E) Inhibition of tumor growth. Note: Statistical analysis was performed using one-way ANOVA for panels (A) and (B), and repeated-measures two-way ANOVA for panel (C) using GraphPad Prism v 9.5.1. [Figure 11C] Figure 11 shows the co-administration of CD3ε-PepPOL, where v1 reduced cytokine release while maintaining antitumor activity in a preclinical MC38-muCD19 tumor model. (A) Serum cytokine INF-y; (B) Serum cytokine-releasing TNF-a; (C) Target cell depletion in the spleen and (D) in the blood; (E) Inhibition of tumor growth. Note: Statistical analysis was performed using one-way ANOVA for panels (A) and (B), and repeated-measures two-way ANOVA for panel (C) using GraphPad Prism v 9.5.1. [Figure 11D]Figure 11 shows the co-administration of CD3ε-PepPOL, where v1 reduced cytokine release while maintaining antitumor activity in a preclinical MC38-muCD19 tumor model. (A) Serum cytokine INF-y; (B) Serum cytokine-releasing TNF-a; (C) Target cell depletion in the spleen and (D) in the blood; (E) Inhibition of tumor growth. Note: Statistical analysis was performed using one-way ANOVA for panels (A) and (B), and repeated-measures two-way ANOVA for panel (C) using GraphPad Prism v 9.5.1. [Figure 11E] Figure 11 shows the co-administration of CD3ε-PepPOL, where v1 reduced cytokine release while maintaining antitumor activity in a preclinical MC38-muCD19 tumor model. (A) Serum cytokine INF-y; (B) Serum cytokine-releasing TNF-a; (C) Target cell depletion in the spleen and (D) in the blood; (E) Inhibition of tumor growth. Note: Statistical analysis was performed using one-way ANOVA for panels (A) and (B), and repeated-measures two-way ANOVA for panel (C) using GraphPad Prism v 9.5.1. [Figure 12A] Figure 12 shows the simultaneous administration of CD20 TCE + CD3ε-PepPOL, where v1 cleaves cytokine release (CD20 + B cell lysis) from the PD response in cynomolgus monkeys. [Figure 12B] Figure 12 shows the simultaneous administration of CD20 TCE + CD3ε-PepPOL, where v1 cleaves cytokine release (CD20 + B cell lysis) from the PD response in cynomolgus monkeys. [Figure 12C] Figure 12 shows the simultaneous administration of CD20 TCE + CD3ε-PepPOL, where v1 cleaves cytokine release (CD20 + B cell lysis) from the PD response in cynomolgus monkeys. [Figure 12D] Figure 12 shows the simultaneous administration of CD20 TCE + CD3ε-PepPOL, where v1 cleaves cytokine release (CD20 + B cell lysis) from the PD response in cynomolgus monkeys. [Figure 12E]Figure 12 shows the simultaneous administration of CD20 TCE + CD3ε-PepPOL, where v1 cleaves cytokine release (CD20 + B cell lysis) from the PD response in cynomolgus monkeys. [Figure 13] Figure 13 shows the clinical chemistry parameters after co-administration of CD20 TCE + antiaffinity CD3ε-PepPOL (pGlu, 20kDa). [Figure 14A] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 14B] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 14C] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 14D] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 14E] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 14F] Figure 14 shows that co-administration of CD20 TCE + high-affinity CD3ε-PepPOL (pGlu, 20kDa) reduces cytokine release in cynomolgus monkeys without affecting the PD effect. [Figure 15A]Figure 15 shows the cytotoxic inhibition of heterodimer STEAP1xCD3 TCE (containing CD3e binder SEQ ID NO: 381), CLDN6xCD3 TCE (containing CD3e binder I2E, SEQ ID NO: 383), and DLL3xCD3 TCE (containing CD33 binder I2C, SEQ ID NO: 26) by masking CD3e. CD3e pepPEG, which has high affinity for the CD3e binder, showed higher inhibitory activity against TCE, while peptides with moderate affinity showed lower inhibitory activity. All high-affinity peptides showed similar efficacy against each other. The masking molecules identified as 79674-1 (20kDa 4-arm PEG and thiosuccinimide linker) and 79675-1 (5kDa 4-arm PEG and thiosuccinimide linker) showed the greatest inhibitory activity in vitro. [Figure 15B] Figure 15 shows the cytotoxic inhibition of heterodimer STEAP1xCD3 TCE (containing CD3e binder SEQ ID NO: 381), CLDN6xCD3 TCE (containing CD3e binder I2E, SEQ ID NO: 383), and DLL3xCD3 TCE (containing CD33 binder I2C, SEQ ID NO: 26) by masking CD3e. CD3e pepPEG, which has high affinity for the CD3e binder, showed higher inhibitory activity against TCE, while peptides with moderate affinity showed lower inhibitory activity. All high-affinity peptides showed similar efficacy against each other. The masking molecules identified as 79674-1 (20kDa 4-arm PEG and thiosuccinimide linker) and 79675-1 (5kDa 4-arm PEG and thiosuccinimide linker) showed the greatest inhibitory activity in vitro. [Figure 15C]Figure 15 shows the inhibition of cytotoxicity of heterodimeric STEAP1xCD3 TCE (including CD3e binder SEQ ID NO: 381), CLDN6xCD3 TCE (CD3e binder I2E, including SEQ ID NO: 383), and DLL3xCD3 TCE (CD33 binder I2C, including SEQ ID NO: 26) by masking CD3e. CD3e pepPEG having high affinity for the CD3e binder showed higher inhibitory activity against the TCE, while peptides with moderate affinity showed lower inhibitory activity. All peptides with high affinity showed similar efficacy against each other. Masking molecules identified as 79674-1 (20 kDa 4-arm PEG and thiol succinimide linker) and 79675-1 (5 kDa 4-arm PEG and thiol succinimide linker) showed the maximum inhibitory activity in vitro. [Figure 16] Figure 16 shows the inhibition of cytokine release, showing the inhibition of cytokine release by a representative TCE masking molecule induced by heterodimeric STEAP1xCD3ε TCE. [Figure 17] Figure 17 shows the inhibition of cytokine release, showing the inhibition of cytokine release by a representative TCE masking molecule induced by DLL3xCD3ε TCE. [Figure 18] Figure 18 shows the inhibition of cytokine release, showing the inhibition of cytokine release by a representative TCE masking molecule induced by CLDN6xCD3ε TCE. [Figure 19] Figure 19 shows the affinity of a representative binding peptide and one negative example in FLT3xCD3 TCE and STEAP1xCD3 TCE. [Figure 20] Figure 20 shows the inhibition of cytotoxicity in CDH3xMSLN dual-targeted TCE. [Figure 21] Figure 21 shows the inhibition of cytokine release in CDH3xMSN dual-targeted TCE.

BEST MODE FOR CARRYING OUT THE INVENTION

[0085] While not limited by theory, the release of pro-inflammatory cytokines, such as those in CRS, is considered a phenomenon rooted in suboptimal TCE pharmacokinetics, in relation to high TCE therapeutic efficacy. Surprisingly, it has been found that when combined with a masking molecule that transiently binds to the T-cell engaging paratope of TCE, thereby transiently masking TCE, a comparable degree of control over CRS incidence can be achieved during a standard IV bolus administration of TCE. Figure 1 illustrates, for example, the general principle of the fundamental pharmacokinetic and pharmacodynamic relationship between TCE exposure—uncontrolled and controlled—by masking molecules and cytokine release. The masking molecule of the present invention generally comprises a peptide that binds to the T-cell engaging paratope of TCE, a linker, and a half-life-extending polymer. Preferred paratopes of the CD3-binding domain of TCE include those that bind to CD3δ, CD3ε, or both; however, CD3ε is used in the context of this disclosure. The masking molecule may be abbreviated as CD3 PepPOL in the following: The abbreviated term CD3ε PepPOL refers to a masking molecule that binds to the CD3ε-binding paratope of TCE. By combining CD3 PepPOL with TCE in a specified molar ratio, a slow PK profile of "free" or "active" TCE can be achieved without the inconvenience of multiple dosing stages or long IV infusion cycles required for drug administration in hospitalized patients.

[0086] Part of the benefits of this disclosure are (i) reducing the frequency of CRS incidence at all TCE dose levels, (ii) facilitating clinical dosing plans by enabling higher initial doses of TCE, and (iii) reducing the number of dose splitting stages of TCE required to achieve an effective dose and thereby shorten the treatment cycle time.

[0087] Advantageously, the masking molecule CD3 PepPOL, when combined with other therapies such as TCE administration before, during, or after TCE administration (e.g., IV bolus or sc administration in TCE monotherapy), can achieve a more favorable TCE exposure profile than TCE administration without the masking molecule. While we do not wish to be bound by theory, this is due to the combination of high affinity / specific binding of CD3 PepPOL to the TCE anti-CD3ε engager moiety and the relatively short lifespan of the CD3 PepPOL PK profile. While we do not wish to be bound by theory, the peptide bond portion of CD3 PepPOL originates from individual CD3 domains of the T cell receptor (TCR) complex, such as the CD3δ domain, CD3ε domain, or a combination thereof, and therefore binds to the anti-CD3 T cell engager moiety with medium to high affinity antagonism to prevent T cell engagement. Thus, as long as a significant amount of CD3 PepPOL remains in circulation compared to the blood concentration of TCE, engagement in the immune system is generally prevented. Importantly, since CD3 PepPOL is generally removed from plasma faster than TCE, competitive blockade of TCE in T cells is transient, and its duration is determined by CD3 PepPOL clearance (CL) and volume of distribution (V), as well as the amount of molar excess CD3 PepPOL administered. For illustrative purposes, if TCE exposure is monitored in terms of active or "free" therapeutic exposure (i.e., TCE exhibiting complete binding complement to tumor antigen and TCR), a PK profile is predicted characterized by a decrease in maximum concentration (Cmax) along with a delay time (tmax) of maximum exposure. The general concept is illustrated through modeling and simulation in Figure 2.

[0088] Generally, the binding peptides of this disclosure are preferably more affinity to the CD3ε paratope of the TCE to be masked than the physiological CD3ε epitope (e.g., sequence containing QDGNEEMG, SEQ ID NO: 262), in terms of a lower Kd value, preferably an equilibrium Kd value. Generally, the Kd value of a preferred binding peptide is, for example, at least 1.5 lower, preferably at least 2 times lower, more preferably 3 times lower, or even about 5 times, about 10 times, or about 100 times lower than the reference epitope QDGNEEMG (SEQ ID NO: 262). See Example 1, for example, SEQ ID NO: 262 shows a binding affinity of 35.8 nM to FLT3xCD3ε TCE (SEQ ID NO: 93). Particularly preferred are any of the binding peptides of SEQ ID NOs: 300, 304-307, 384, and 389. The use of high-affinity binding peptides generally allows for a lower molar ratio of masing molecule to TCE, thus making the use of masking molecules more economical.

[0089] In particular, binding peptides that result in improved affinity through the substitution of the N-terminal amino acid Q with pE are preferred. Affinity can be further fine-tuned, in particular, by the amino acid at position 4 in the N-to-C direction of the binding peptide according to the present invention. Amino acid N is generally associated with higher affinity, while amino acid E is associated with slightly attenuated, i.e., moderate affinity. Moderate affinity may be advantageous for promoting relatively short-term masking of TCE. A key problem to be solved by the present invention is to mitigate cytokine release induced by TCE treatment. As described herein, and without wishing to be bound by theory, such cytokine release may be accompanied by initial high systemic TCE exposure, for example, in plasma. Therefore, transient masking of TCE is sufficient to mitigate cytokine release at the initiation of TCE treatment. The masking time is generally determined by the affinity of the CD3ε paratope and the stability of the TCE masking molecule. In this view, binding peptides with half-lives of less than approximately 24 hours, preferably about 12, 10, 8, 6, 4, or 2 hours, are advantageous for mitigating initial high systemic exposure to TCE, but do not reduce TCE exposure for longer than necessary to mitigate initial cytokine release in order to ensure TCE-on-target activity. Generally, the half-lives of TCEs containing half-life-extending domains, such as Fc-based domains, are typically greater than 24 hours, preferably more than 2 days. The half-life of the binding peptide alone is only a few minutes, as shown in the examples, which is too short to effectively mitigate initial TCE-mediated cytokine release. Half-life is generally understood herein as the half-life in serum.

[0090] In general, all polymers disclosed herein are suitable for linking to binding peptides, such as those disclosed herein, via linkers, such as those disclosed herein, in order to extend the half-life of the binding peptide. Peptides that have been proven clinically safe and approved by regulatory authorities are preferred. In this respect, PEG and PLGA are preferred. Interestingly, branching of polymers may provide further advantages for TCEs having multiple CD3ε domains. For example, a dual-target CDH3xMSLN molecule having two CD3ε domains, such as SEQ ID NO: 255, can be masked more efficiently by a masking molecule containing 4-armed PEG than by linear PEG.

[0091] In relation to this disclosure, a binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of a TCE with an affinity expressed as Kd of approximately 0.05 to approximately 10 nM, generally 0.1 pM to 10 nM or 500 pM to 5 nM, is considered to have "high" affinity, and a binding peptide that binds to the anti-CD3ε paratope of the CD3ε-binding domain of a TCE with an affinity of approximately 10 to 150, preferably 10 to 100 mM, is also considered to have "high" affinity. The exact values ​​may vary in individual TCEs. However, the

[0092] (i.) The binding peptide is preferably one that includes an N-terminal pE to preferably enhance affinity with respect to the physiological epitope containing QDGNEEM to which the anti-CD3ε paratope of the CD3ε binder of TCE binds, (ii.) preferably an E or N located at position 4 in the N-to-C direction to fine-tune high or moderate affinity, (iii.) an E at position 5 in the E-to-N direction, and (iv.) preferably an N-terminal C or K to preferably provide efficient linker linkage. For example, pEX1X2X3EX4X5X6GX7 (SEQ ID NO: 433) (where X1 is D, H, or N, preferably D; X2 is G, F, or Y, preferably G; X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N or E; X4 is E, L, P, or W, preferably E; and X5 is any proteogenic amino acid, i.e., A, C The expression is for 10-mer linked peptides such as pEX1X2X3EEX5X6GX7, more preferably pEDGX3EEX5X6GX7.

[0093] In general, the following findings exist regarding the anti-CD3ε paratope-binding peptide of this disclosure; see Table 3.

[0094] [Table 11]

[0095] In the context of this disclosure, the term “binding peptide” refers to a chain of about 5 to about 20 amino acids, preferably about 6 to about 12 amino acids, for example, 10 amino acids, that binds to the anti-CD3ε paratope of the CD3ε-binding domain of TCE. The amino acid chain may include proteogenic and non-proteogenic amino acids. A preferred non-proteogenic amino acid is pyroglutamic acid (pE), also known as 5-oxoproline.

[0096] The term "CD3" refers to "surface antigen classification 3," a T cell co-receptor involved in the activation of cytotoxic CD8+ T cells, for example. Co-receptors are generally known as cell surface receptors that bind to signaling molecules in addition to the primary receptor to facilitate ligand recognition and initiate biological processes. CD3-epsilon, along with CD3-gamma, -delta, and -zeta and T cell receptor alpha / beta and gamma / delta heterodimers, forms the T cell receptor-CD3 complex. This complex plays a crucial role in linking antigen recognition to several intracellular signaling pathways.

[0097] The term "TCE masking molecule" refers to a molecule comprising an anti-CD3 paratope-binding peptide, preferably an anti-CD3ε-paratope-binding peptide, a suitable linker, and a suitable polymer that extends the half-life of the TCE masking molecule compared to the binding peptide alone. The TCE masking molecule may also be referred to herein interchangeably as a CD3 peptide polymer (CD3 pepPOL) or more specifically as CD3 pepPEG.

[0098] The term “antibody product” refers to “secreted protein” or “secreted recombinant protein,” meaning a protein (e.g., recombinant protein) that originally contains at least one secretory signal sequence when translated within a mammalian cell and is secreted at least partially into the extracellular space (e.g., liquid culture medium) via enzymatic cleavage of the secretory signal sequence within the mammalian cell. Those skilled in the art will understand that a “secreted” protein does not need to be completely dissociated from the cell to be considered a secreted protein.

[0099] The term "bispecific antibody" refers to full-length bispecific antibodies, such as IgG-based antibodies. On the other hand, a bispecific antibody fragment is a part of a full-length antibody that has a specified function, and both are colloquially referred to as bispecific antigen-binding molecules in this specification.

[0100] The term “T cell engager” refers to a bispecific antigen-binding molecule comprising at least one binding domain that binds to an antigen or target (e.g., a target cell surface antigen, preferably a tumor-associated antigen (TAA)) and a second binding domain that binds to another antigen or target, in this context CD3, preferably CD3ε. Unless otherwise indicated, all references to CD3 in this disclosure refer to CD3ε. CD3ε may also be written as CD3e and refer to the same subject. TCE is understood to mean an “antigen-binding molecule” whose structure and / or function are based on the structure and / or function of an antibody, e.g., the entire full-length or immunoglobulin molecule, and / or derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or its fragment. Thus, an antigen-binding molecule binds to its specific target or antigen. Furthermore, the binding domain of the antigen-binding molecule according to the present invention includes the minimum structural requirements of the antibody that enable target binding. This minimum requirement can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), preferably all six CDRs. Alternative methods for defining the minimum structural requirements of an antibody are by defining the structure of a specific target, defining the antibody's epitope within the protein domains (epitope clusters) of the target protein that constitute the epitope region, or by referring to a specific antibody that competes with the epitope of the defined antibody. Antibodies on which the construct according to the present invention is based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0101] The binding domain of the antigen-binding molecule according to the present invention may include, for example, the CDRs of the groups referenced above. Preferably, these CDRs are included within the frameworks of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but it is not necessary to include both. The Fd fragment has, for example, two VH regions and often retains the antigen-binding function of a part of the intact antigen-binding domain. Further examples of forms of antibody fragments, antibody variants or binding domains include: (1) the Fab fragment, which is a monovalent fragment having VL, VH, CL and CH1 domains; (2) the F(ab’)2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; (3) the Fd fragment having two VH and CH1 domains; (4) the Fv fragment having the VL and VH domains of one arm of the antibody; (5) the dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) the isolated complementarity-determining region (CDR), and (7) the single-chain Fv (scFv), with the latter being preferred (for example, those derived from scFV libraries). Examples of embodiments of the antigen-binding molecule according to the present invention are described, for example, in WO 00 / 006605 pamphlet, WO 2005 / 040220 pamphlet, WO 2008 / 119567 pamphlet, WO 2010 / 037838 pamphlet, WO 2013 / 026837 pamphlet, WO 2013 / 026833 pamphlet, US Patent Application Publication No. 2014 / 0308285 specification, US Patent Application Publication No. 2014 / 0302037 specification, WO 2014 / 144722 pamphlet, WO 2014 / 151910 pamphlet and WO 2015 / 048272 pamphlet.

[0102] Furthermore, the definition of “binding domain” or “domain that binds” also includes fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or “r IgG” (“half-antibody”). The antigen-binding molecules according to the present invention may also include modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, “multibodies,” such as triabodies or tetrabodies, and single-domain antibodies, such as nanobodies, or single variable-domain antibodies containing only one variable domain that may be VHH, VH, or VL that specifically binds to an antigen or epitope independent of other V regions or domains.

[0103] As used herein, the terms “single-chain Fv,” “single-chain antibody,” or “scFv” refer to an antibody fragment of a single polypeptide chain that contains variable regions derived from both the heavy and light chains but lacks a constant region. Typically, a single-chain antibody further includes a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that allows binding to an antigen. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods for producing single-chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Publication No. 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; and Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single-chain antibodies may be bispecific, multispecific, human and / or humanized and / or synthetic.

[0104] Furthermore, the definition of the term "antigen-binding molecule" includes monovalent, divalent, and polyvalent / multivalent constructs, thus bispecific constructs that specifically bind to only two antigenic structures, as well as polyspecific / multispecific constructs that specifically bind to three or more antigenic structures, e.g., three, four, or more, through different binding domains. In addition, the definition of the term "antigen-binding molecule" includes molecules consisting of only one polypeptide chain, and molecules consisting of multiple polypeptide chains (these chains may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers)). Examples of the antibodies and variants or derivatives identified above are described, in particular, in Harlow and Lane, Antibodies: A Laboratory Manual, CSHL Press (1988) and Using Antibodies: A Laboratory Manual, CSHL Press (1999), Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0105] The term "polypeptide," as used herein, refers to a group of molecules typically consisting of more than 30 amino acids. Polypeptides may further form multimers, such as dimers, trimers, and higher-order oligomers, i.e., they may consist of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc., may or may not be identical. The corresponding higher-order structures of such multimers are therefore referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its natural form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally occurring modified peptides / polypeptides / proteins that are modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation. "Peptides," "polypeptides," or "proteins," as referred herein, may also be chemically modified, such as pegylation. Such modifications are well known in the art and are described below herein.

[0106] As used herein, the term “bispecific” means an antigen-binding molecule that is “at least bispecific,” i.e., it comprises at least a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (e.g., a surface antigen of a target cell), and the second binding domain binds to another antigen or target. In the context of this disclosure, one of the two binding domains of a bispecific antigen-binding molecule binds to an extracellular epitope of CD3, preferably CD3ε, more preferably CD3ε. Since a bispecific antigen-binding molecule as defined above can engage T cells, the term bispecific antigen-binding molecule is used herein interchangeably with T cell engager (TCE) or TCE molecule. A TCE having two CD3ε binding domains in addition to at least one target-binding domain is also considered bispecific. However, a TCE containing at least two target-binding domains in addition to at least one CD3ε binding domain is understood to be bitargetable in addition to being bispecific. Thus, the antigen-binding molecules according to the present invention include specificity for at least two different antigens or targets. For example, the first domain preferably does not bind to one or more extracellular epitopes of species CD3ε as described herein. The term “target cell surface antigen” refers to an antigenic structure expressed by a cell and present on the cell surface so that an antigen-binding molecule as described herein can reach it. This may be a protein (preferably the extracellular portion of a protein) or a carbohydrate structure (preferably a carbohydrate structure of a protein such as a glycoprotein). This is preferably a tumor antigen. The term “bispecific antigen-binding molecule” of the present invention also includes triplespecific antigen-binding molecules containing three binding domains, or multispecific antigen-binding molecules such as constructs having more than three (e.g., four, five...) specificities.

[0107] If the antigen-binding molecule according to the present invention is (at least) bispecific, it does not exist in nature and is significantly different from naturally occurring products. Therefore, a “bispecific” antigen-binding molecule or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sites with different specificities. Bispecific antigen-binding molecules can be produced by various methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0108] At least two binding domains and variable domains (VH / VL) of the antigen-binding molecule of the present invention may or may not contain a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" includes an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antigen-binding molecule of the present invention to each other. This peptide linker may also be used to fuse a third domain to other domains of the antigen-binding molecule of the present invention. An essential technical feature of such peptide linkers is that they do not contain polymerization activity. Suitable peptide linkers are described in U.S. Patent Nos. 4,751,180 and 4,935,233 or International Publication No. 88 / 09344. Peptide linkers may also be used to attach other domains, modules, or regions (such as half-life extension domains) to the antigen-binding molecule of the present invention.

[0109] The antigen-binding molecule of the present invention is preferably an "in vitro-generated antigen-binding molecule." This term refers to an antigen-binding molecule as defined above, in which all or part of the variable region (e.g., at least one CDR) is generated in any other way that allows for the selection of non-immune cells, e.g., in vitro phage display, protein chip, or testing of candidate sequences for antigen-binding ability. Accordingly, this term preferably excludes sequences generated solely by genomic rearrangement in animal immune cells. A "recombinant antibody" is an antibody produced by the use of recombinant DNA technology or genetic engineering.

[0110] The term “monoclonal antibody” (mAb) or “monoclonal antigen-binding molecule,” as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies that are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are induced to a single antigenic site or determinant on an antigen, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies induced to different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore not contaminated by other immunoglobulins. The modifier “monoclonal” indicates an antibody characteristic such as that obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.

[0111] For the preparation of monoclonal antibodies, any technique resulting from antibody production by continuous cell line culture may be used. For example, the monoclonal antibody to be used may be produced by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Further examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0112] Next, hybridomas can be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE®) analysis to identify one or more hybridomas that produce antibodies that specifically bind to a specified antigen. Any form of the relevant antigen can be used as an immunogen, for example, recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptide. Using surface plasmon resonance employed in the BIAcore system, the efficiency of phage antibody binding to epitopes of surface antigens on target cells can be increased (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J.Immunol.Methods 183(1995), 7-13).

[0113] Another exemplary method for producing monoclonal antibodies involves screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985), Science 228:1315-1317; Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J.Mol.Biol., 222:581-597 (1991).

[0114] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (e.g., mice, hamsters, rabbits, or rats). In one embodiment, the non-human animal contains at least a portion of the human immunoglobulin gene. For example, a mouse strain lacking the production of mouse antibodies can be modified using a large fragment of the human Ig (immunoglobulin) locus. Hybridoma technology can be used to produce and select antigen-specific monoclonal antibodies derived from genes with desired specificity. See, for example, XENOMOUSE®, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, and International Publications No. 96 / 34096 and 96 / 33735.

[0115] Monoclonal antibodies can also be obtained from non-human animals and then modified, for example, by humanization, deimmunization, chimerization, etc., using recombinant DNA techniques known in the art. Examples of modified antigen-binding molecules include humanized variants of non-human antibodies, "affinity-mature" antibodies (e.g., Hawkins et al. J.Mol.Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)) and antibody mutations with altered effector function (e.g., U.S. Patent Application No. 5,648,260, Kontermann and Dubel (2010), op. cit. and Little (2009), op. cit.).

[0116] In immunology, affinity maturation is the process by which B cells produce antibodies with increased affinity to an antigen during an immune response. Repeated exposure to the same antigen causes the host to continuously produce antibodies with higher affinity. Similar to natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antigen-binding molecules, and antibody fragments. Random mutations within the CDR are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, chain shuffling can increase genetic diversity. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinities in the low nanomolar range.

[0117] A preferred type of amino acid substitution variant of an antigen-binding molecule involves substitution of one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variants, selected for further development, have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are then presented in a monovalent form from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites to modify, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystalline structure of the antigen-antibody complex to identify the contact sites between the binding domain and, for example, the surface antigen of a human target cell. Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein. After generating such variants, a panel of variants may be screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0118] The monoclonal antibodies and antigen-binding molecules of the present invention include, in particular, antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain is derived from a different species or belonging to a different class or subclass of antibodies, insofar as it exhibits the desired biological activity, as well as “chimeric” antibodies (immunoglobulins) that are identical or homologous to a corresponding sequence in a fragment of such an antibody (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest as used herein include “primatized” antibodies that include a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence. Various methods for producing chimeric antibodies are described. For example, see Morrison et al., Proc. Natl. Acad. Sci USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent No. 0171496; European Patent No. 0173494; and British Patent No. 2177096.

[0119] Antibodies, antigen-binding molecules, antibody fragments, or antibody variants can also be modified by specific deletion of human T cell epitopes (a method called "deimmunization"), for example, by the methods disclosed in International Publication No. 98 / 52976 or International Publication No. 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed to determine whether they are peptides that bind to MHC class II, and these peptides indicate that they are potential T cell epitopes (as defined in International Publication No. 98 / 52976 and International Publication No. 00 / 34317). To detect potential T cell epitopes, a computer modeling technique called "peptide threading" can be applied, as described in International Publication No. 98 / 52976 and International Publication No. 00 / 34317, and in addition, a database of human MHC class II-binding peptides can be searched for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes and thus become potential T cell epitopes. Detected potential T cell epitopes can be eliminated by substituting a few amino acid residues in the variable domain, or preferably by substituting a single amino acid. Typically, conservative substitutions are performed. In many, though not all, amino acids common to the positions in human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J.MoI. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16(5):237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used, for example, as a source of human sequences for framework regions and CDRs.For example, a consensus human framework area, such as that described in U.S. Patent No. 6,300,064, can also be used.

[0120] A “humanized” antibody, antigen-binding molecule, variant, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) is an antibody or immunoglobulin that is predominantly human, containing minimal sequences derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s hypervariable region (also known as the CDR) are replaced with residues from the hypervariable region of a non-human species (e.g., rodent) such as mouse, rat, hamster, or rabbit (donor antibody) having the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, “humanized antibody,” as used herein, may also include residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize the performance of the antibody. A humanized antibody may also include the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0121] Humanized antibodies or fragments thereof can be prepared by substituting the sequence of the Fv variable domain, which is not directly involved in antigen binding, with an equivalent sequence derived from the human Fv variable domain. Exemplary methods for preparing humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; and U.S. Patents No. 5,585,089; No. 5,693,761; No. 5,693,762; No. 5,859,205 and No. 6,407,213. These methods involve the isolation, manipulation, and expression of nucleic acid sequences encoding all or part of the immunoglobulin Fv variable domain derived from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources that produce antibodies against a given target as described above. Next, recombinant DNA encoding a humanized antibody molecule can be cloned into a suitable expression vector.

[0122] Humanized antibodies can also be produced using transgenic animals, such as mice that express human heavy and light chain genes but cannot express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes exemplary CDR transplantation methods that can be used for the preparation of humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least some of the non-human CDRs, or only some of the CDRs may be replaced with non-human CDRs. Only the number of CDRs required for the binding of the humanized antibody to a given antigen needs to be replaced.

[0123] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or reverse mutations. Such modified immunoglobulin molecules can be prepared by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982; and European Patent No. 239400).

[0124] The terms “human antibody,” “human antigen-binding molecule,” and “human binding domain” include antibodies, antibody-binding molecules, and binding domains having antibody regions such as variable regions and constant regions or domains that substantially correspond to human germline immunoglobulin sequences known in the art, including those described in Kabat et al. (1991) (cited above). The human antibody, antigen-binding molecule, or binding domain of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or in vivo somatic mutation), for example, in CDR, particularly CDR3. The human antibody, antigen-binding molecule, or binding domain may have at least one, two, three, four, five, or more positions replaced by amino acid residues not encoded by human germline immunoglobulin sequences. However, as used herein, the definitions of human antibody, antigen-binding molecule, and binding domain can be obtained using techniques or systems such as Xenomouse, and therefore, “fully human antibody” also refers to an antibody containing only the human sequence of a non-artificial and / or genetically modified antibody. Preferably, a “fully human antibody” does not contain amino acid residues not encoded by a human germline immunoglobulin sequence.

[0125] In some embodiments, the antigen-binding molecules of the present invention are “isolated” or “substantially pure” antigen-binding molecules. When “isolated” or “substantially pure” is used in the description of the antigen-binding molecules disclosed herein, it means antigen-binding molecules identified, separated and / or recovered from components of their production environment. Preferably, the antigen-binding molecules do not associate with or substantially associate with all other components from their production environment. Contaminating components of their production environment, such as components arising from recombinant transfected cells, are typically materials that interfere with diagnostic or therapeutic applications relating to polypeptides and may include enzymes, hormones, and other proteolytic or non-proteolytic solutes. The antigen-binding molecules may constitute, for example, at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that isolated proteins may constitute 5% to 99.9% by weight of the total protein content, depending on the context. Polypeptides can be produced at significantly higher concentrations by using inducible promoters or high-expression promoters so that they are produced at increased concentration levels. This definition includes the generation of antigen-binding molecules in a wide variety of organisms and / or host cells known in the art. In preferred embodiments, the antigen-binding molecule is purified (1) by using a spinning cup sequencer to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (2) by SDS-PAGE under non-reducible or reducing conditions using Coomassie blue or preferably silver staining until homogeneous. However, typically, the isolated antigen-binding molecule is prepared by at least one purification step.

[0126] In relation to the present invention, the term "binding domain" is considered to be a domain that (specifically) binds to / interacts with / recognizes a given target epitope or target site on a target molecule (antigen), for example, CD33 and CD3, respectively. The structure and function of the first binding domain (e.g., CD33 recognition), preferably the structure and / or function of the second binding domain (e.g., CD3 recognition), are also based on the structure and / or function of the antibody, for example, the full-length or full immunoglobulin molecule, and / or extracted from the variable heavy chain (VH) and / or variable light chain (VL) domains of the antibody or its fragments. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The second binding domain preferably also includes the minimum structural requirements of the antibody that enable target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The first and / or second binding domains are expected to be prepared or obtained by phage display or library screening methods other than transplanting CDR sequences derived from existing (monoclonal) antibodies onto a backbone.

[0127] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may comprise a protein portion and a non-protein portion (e.g., a chemical linker or a chemical crosslinking agent such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments and peptides having typically fewer than 30 amino acids) comprise two or more amino acids linked to one another via covalent peptide bonds (resulting in a chain of amino acids).

[0128] As used herein, the term “polypeptide” typically refers to a group of molecules consisting of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers, and higher-order oligomers, i.e., they may consist of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc., may or may not be identical. The corresponding higher-order structures of such multimers are therefore referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule that, in its natural form, consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms “peptide,” “polypeptide,” and “protein” also refer to naturally occurring modified peptides / polypeptides / proteins that have been modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation. “Peptides,” “polypeptides,” or “proteins,” as referred herein, may also be chemically modified, such as pegylated. Such modifications are known in the art and are described below herein.

[0129] Preferably, the binding domain that binds to the surface antigen of the target cell and / or the binding domain that binds to CD3ε is a human binding domain. Antibodies and antigen-binding molecules containing at least one human binding domain avoid some of the problems associated with antibodies or antigen-binding molecules that have non-human variable regions and / or constant regions, such as those from rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins can lead to rapid clearance of the antibody or antigen-binding molecule or to an immune response by the patient to the antibody or antigen-binding molecule. To avoid using rodent-derived antibodies or antigen-binding molecules, human or fully human antibody / antigen-binding molecules can be produced by introducing human antibody function into rodents so that the rodents produce fully human antibodies.

[0130] The ability of YAC to clone and reconstruct megabase-sized human loci and to introduce them into mouse germline cells provides a powerful method for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human diseases. Furthermore, using such techniques to replace mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their transduction to other systems, and their involvement in disease induction and progression.

[0131] A key practical application of such strategies is the "humanization" of the mouse humoral immune system. Introducing human Ig loci into mice with inactivated endogenous immunoglobulin (Ig) genes provides an opportunity to study the mechanisms underlying programmed antibody expression and construction, as well as their roles in B cell development. Furthermore, such strategies could provide an ideal source for producing fully human monoclonal antibodies (mAbs), a crucial milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antigen-binding molecules are expected to minimize the immunogenicity and allergic reactions inherent in mouse mAbs or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibody / antigen-binding molecules. The use of fully human antibodies or antigen-binding molecules is expected to offer significant advantages in the treatment of chronic and recurrent human diseases requiring repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0132] One approach to this goal involves modifying mouse strains lacking mouse antibody production with a large fragment of the human Ig locus, based on the prediction that such mice would produce a broad repertoire of human antibodies in the absence of mouse antibodies. The large human Ig fragment is thought to retain broad diversity of variable genes as well as appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection, and for the lack of immune tolerance to human proteins, the human antibody repertoire reproduced in these mouse strains should produce high-affinity antibodies against any target antigen, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity can be easily generated and selected. This general strategy was demonstrated in connection with the creation of the first XenoMouse mouse strain (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered using yeast artificial chromosomes (YACs) containing germline-arranged fragments of 245kb and 190kb sizes, respectively, of the human heavy chain locus and kappa light chain locus, containing core sequences of the variable and constant regions. These human Ig-containing YACs proved compatible with the mouse strain for both antibody rearrangement and expression, and were capable of replacing inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing numerous V genes, additional regulatory elements, and the human Ig constant region, could reproduce a substantially complete repertoire characterized by the human humoral response to infection and immunization. More recently, building upon the work of Green et al., the introduction of megabase-sized germline-arranged YAC fragments of the human heavy chain locus and kappa light chain locus resulted in the introduction of over 80% of the human antibody repertoire.See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application No. 08 / 759,620.

[0133] Regarding the production of the XenoMouse mouse, U.S. Patent Applications No. 07 / 466,008, No. 07 / 610,515, No. 07 / 919,297, No. 07 / 922,649, No. 08 / 031,801, No. 08 / 112,848, No. 08 / 234,145, No. 08 / 376,279, No. 08 / 430,938, No. 08 / 464,584, No. 08 / 464,582, No. 08 / 463, Further discussion and detail are provided in Patent No. 191, No. 08 / 462,837, No. 08 / 486,853, No. 08 / 486,857, No. 08 / 486,859, No. 08 / 462,513, No. 08 / 724,752 and No. 08 / 759,620; and U.S. Patent No. 6,162,963, No. 6,150,584, No. 6,114,598, No. 6,075,181 and No. 5,939,598, as well as Japanese Patent Publication No. 3068180B2, No. 3068506B2 and No. 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J.Exp.Med.188:483-495 (1998), European Patent No. 0463151B1, International Publication Brochures 94 / 02602, 96 / 34096, 98 / 24893, 00 / 76310, and 03 / 47336.

[0134] An alternative approach, utilizing the "minilocus" method, is employed by other companies, including GenPharm International, Inc. In this minilocus method, an exogenous Ig locus is mimicked by including fragments (individual genes) derived from this Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a muon constant region, and a second constant region (preferably a gamma constant region) form a construct that is inserted into the animal. This approach applies to U.S. Patent No. 5,545,807 to Surani et al., and to U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 to Krimpenfort and Berns, and to Berns et al. U.S. Patent Nos. 5,612,205; 5,721,367; and 5,789,215 for al., and U.S. Patent Nos. 5,643,763 for Choi and Dunn, and GenPharm This is described in U.S. Patent Applications No. 07 / 574,748, No. 07 / 575,962, No. 07 / 810,279, No. 07 / 853,408, No. 07 / 904,068, No. 07 / 990,860, No. 08 / 053,131, No. 08 / 096,762, No. 08 / 155,301, No. 08 / 161,739, No. 08 / 165,699, and No. 08 / 209,741 filed by International.It is also described in European Patent No. 0546073B1, International Publication Nos. 92 / 03918, 92 / 22645, 92 / 22647, 92 / 22670, 93 / 12227, 94 / 00569, 94 / 25585, 96 / 14436, 97 / 13852, and 98 / 24884, as well as in U.S. Patent No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), Tuaillon et al. (1995), and Fishwild et al. (1996).

[0135] Kirin has also demonstrated the production of human antibodies from mice in which large chromosome fragments or entire chromosomes are introduced via microcell fusion. See European Patent Publications No. 773288 and 843961. Xenerex Biosciences is developing a technology for generating promising human antibodies. This technology involves reconstituting SCID mice with human lymphocytes, such as B cells and / or T cells. The mice are then immunized with an antigen to induce an immune response to the antigen. See U.S. Patents No. 5,476,996, 5,698,767 and 5,958,765.

[0136] Human-anti-mouse antibody (HAMA) reactions have been a driving force behind the industry's development of chimeric antibodies or other methods of humanized antibody preparation. However, certain human-anti-chimeric antibody (HACA) reactions are expected to be observed, particularly in chronic or multi-dose antibody applications. Therefore, to eliminate concerns and / or effects of HAMA or HACA reactions, it is desirable to provide antigen-binding molecules containing human-binding domains to target cell surface antigens and human-binding domains to CD3ε.

[0137] The terms "(specifically) bind," "(specifically) recognize," "(specifically) induce," and "(specifically) react" mean, according to the present invention, that the binding domain interacts with or specifically interacts with a target molecule (antigen), in this specification, with a surface antigen of a target cell and a given epitope or target site on CD3ε, respectively.

[0138] The term "epitope" refers to a site on an antigen to which a binding domain of an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, binds. An "epitope" is antigenic, and therefore, the term epitope may also be referred to as an "antigenic structure" or "antigenic determinant" in this specification. Thus, the binding domain is an "antigen interaction site." This binding / interaction is understood to also define "specific recognition."

[0139] An "epitope" can be formed by both consecutive amino acids or discontinuous amino acids that are paralleled by the three-dimensional folding of a protein. A "linear epitope" is an epitope that contains an epitope in which the primary amino acid sequence is recognized. Linear epitopes typically contain at least three or at least four and more commonly at least five, at least six, or at least seven amino acids within a characteristic sequence, for example, about 8 to about 10 or 5 or 27 amino acids.

[0140] A "structural epitope," in contrast to a linear epitope, is an epitope where the primary sequence of amino acids containing the epitope is not the sole defining element of the recognized epitope (for example, an epitope where the primary sequence of amino acids is not necessarily recognized by the binding domain). Generally, structural epitopes contain a larger number of amino acids compared to linear epitopes. In relation to the recognition of structural epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein, or a fragment thereof (in relation to the present invention, the antigenic structure for one of the binding domains is contained within the surface antigen protein of the target cell). For example, when a protein molecule folds to form a three-dimensional structure, specific amino acids and / or polypeptide backbone that form the structural epitope are arranged in parallel, thereby enabling the antibody to recognize that epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, site-specific spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0141] The epitope mapping method is described below. When a region (a sequence of adjacent amino acids) of a human target cell's surface antigen protein is exchanged / substituted with the corresponding region of a non-human and non-primate target cell's surface antigen (for example, a mouse target cell's surface antigen, but others such as chicken, rat, hamster, and rabbit are also possible), a reduction in binding affinity of the binding domain is expected to occur, unless the binding domain is cross-reactive to the non-human and non-primate target cell's surface antigen being used. The aforementioned reduction is preferably at least 10%, 20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100%, compared to binding to the corresponding region within the human target cell's surface antigen protein, with binding to the corresponding region of the human target cell's surface antigen protein being 100%. The above human target cell surface antigen / non-human target cell surface antigen chimeras are expected to be expressed in CHO cells. Furthermore, it is conceivable that chimeric forms of surface antigens from human target cells and non-human target cells may fuse with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins, such as EpCAM.

[0142] As an alternative or additional method to epitope mapping, several truncated forms of the extracellular domains of human target cell surface antigens may be created to determine specific regions recognized by the binding domain. In these truncated forms, different extracellular target cell surface antigen domains / subdomains or regions are deleted stepwise, starting from the N-terminus. It is hypothesized that truncated target cell surface antigens may be expressed in CHO cells. It is also hypothesized that truncated target cell surface antigens may be fused with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins, such as EpCAM. Furthermore, it is hypothesized that truncated target cell surface antigens may contain a signal peptide domain at their N-terminus, e.g., a signal peptide derived from mouse IgG heavy chain signal peptide. In addition, it is hypothesized that truncated target cell surface antigens may contain a v5 domain at the N-terminus (following the signal peptide) to confirm their precise expression on the cell surface. In truncated target cell surface antigens that no longer contain the target cell surface antigen region recognized by the binding domain, a reduction or loss of binding is expected. The reduction in binding is preferably at least 10%, 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even further 100%, when the binding to the entire surface antigen protein (or its extracellular region or domain) of the human target cell is set to 100%.

[0143] A further method for determining the contribution of specific residues of a target cell surface antigen to recognition by an antigen-binding molecule or binding domain is alanine scanning (see, for example, Morrison KL & Weiss GA. Curr Opin Chem Biol. 2001 Jun;5(3):302-7), where each residue to be analyzed is replaced with alanine, for example, by site-directed mutagenesis. Alanine is used because, despite mimicking the secondary structure criteria of many other amino acids, it is not bulky and has a chemically inert methyl functional group. If it is desirable to conserve the size of the mutated residue, sometimes bulky amino acids such as valine or leucine may be used. Alanine scanning is a well-established technique that has been used for a long time.

[0144] The interaction between the binding domain and the epitope or region containing the epitope means that the binding domain exhibits a measurable affinity for the epitope / region containing the epitope on a specific protein or antigen (herein, the surface antigen of the target cell and CD3, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than the surface antigen of the target cell or CD3. "Apparent affinity" includes bindings having an affinity of about 10 -6 M (KD) or stronger. Preferably, the binding affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9If M is present, the binding is considered specific. Whether the binding domain specifically reacts with or binds to a target can be easily tested, in particular, by comparing the reaction of the binding domain to the target protein or antigen with the reaction of the binding domain to the surface antigen of the target cell or to proteins or antigens other than CD3. Preferably, the binding domain of the present invention does not essentially or substantially bind to the surface antigen of the target cell or to proteins or antigens other than CD3 (i.e., the first binding domain cannot bind to proteins other than the surface antigen of the target cell, and the second binding domain cannot bind to proteins other than CD3). Having superior affinity properties compared to other HLE forms is a presumed feature of the antigen-binding molecule according to the present invention. Such superior affinity suggests that, as a result, the in vivo half-life is extended. The longer the half-life of the antigen-binding molecule according to the present invention, the less the duration and frequency of administration can be reduced, which typically contributes to improved patient compliance. This is particularly important because the antigen-binding molecule of the present invention is especially beneficial for cancer patients who are severely debilitated or have multiple diseases.

[0145] The term "paratope" is understood as part of an antibody or antibody-derived molecule, such as a TCE, that recognizes and binds to an antigen, i.e., an antigen-binding site. Paratopes are generally understood as small regions within the Fv. Each paratope contains six complementarity-determining regions (CDRs), three each from the light chain and heavy chain. In the context of this invention, a CD3 paratope, or more preferably a CD3ε paratope, facilitates the binding of a TCE molecule to the CD3 receptor in T cells. The CD3 T cell coreceptor is an antigen, more preferably its CD3ε chain, and the paratope of the TCE is a portion of the CD3-binding domain that binds to and contacts the epitope of the antigen.

[0146] The term "anti-CD3ε paratope" is understood as the CD3ε-binding site within the CD3ε-binding domain of a TCE that binds to a corresponding epitope on CD3ε. The anti-CD3ε paratope is preferably the binding site of a binding peptide of a TCE masking molecule to transiently mask the TCE.

[0147] The terms "essentially / substantially non-binding" or "unable to bind" mean that the binding domain of the present invention does not bind to the surface antigen of the target cell or proteins or antigens other than CD3, i.e., if binding to the surface antigen of the target cell or CD3 is taken as 100%, it does not show reactivity to proteins or antigens other than the surface antigen of the target cell or CD3 of the target cell with a reactivity of more than 30%, preferably 20% or less, more preferably 10% or less, and particularly preferably 9%, 8%, 7%, 6%, or 5% or less.

[0148] Binding is thought to be mediated by specific motifs within the binding domain and the amino acid sequence of the antigen. Therefore, binding occurs as a result of their primary, secondary, and / or tertiary structures, as well as as a result of secondary modifications of said structures. Specific interaction between the antigen interaction site and its specific antigen can lead to simple binding of the site to the antigen. Furthermore, specific interaction between the antigen interaction site and its specific antigen can alternatively or additionally initiate a signal, for example, by inducing conformational changes in the antigen or oligomerization of the antigen.

[0149] The term "variable" refers to a portion of an antibody or immunoglobulin domain (i.e., a "variable domain") that exhibits variability within its sequence and is involved in determining the specificity and binding affinity of a particular antibody. A pair of variable heavy chains (VH) and variable light chains (VL) together form a single antigen-binding site.

[0150] The variability is not uniformly distributed throughout the antibody's variable domain, but rather concentrated in the respective subdomains of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs in the three-dimensional space that forms the antigen-binding surface. The naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4), mostly in a β-sheet configuration, which are connected by three hypervariable regions that form loop connections and, in some cases, form part of the β-sheet structure. The hypervariable regions of each chain are held collectively in close proximity by the FRMs and, together with the hypervariable regions from other chains, contribute to the formation of the antigen-binding site (see Kabat et al. cited above).

[0151] The term "CDR" and its plural form "CDRs" refer to complementarity-determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3), and three which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). CDRs contain most of the residues responsible for the specific interaction between antibodies and antigens, and therefore contribute to the functional activity of antibody molecules. CDRs are the main determinants of antigen specificity.

[0152] The precise definition of CDR boundaries and length follows various classification and numbering schemes. Therefore, CDRs may be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering schemes described herein. Even with different boundaries, each of these schemes has some overlap in the portions constituting the so-called "hypervariable regions" within the variable sequence. Consequently, the definitions of CDRs by these schemes may differ in terms of length and the boundary regions with respect to adjacent framework regions. See, for example, Kabat (a method based on interspecies sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., op. cit.; Chothia et al., J.MoI.Biol, 1987, 196:901-917; and MacCallum et al., J.MoI.Biol, 1996, 262:732). Another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Two residue identification techniques can be combined to define hybrid CDRs, insofar as they define overlapping but not identical regions. However, numbering following the so-called Kabat system is preferred.

[0153] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the three-dimensional structure of the main chain adopted by the antigen-binding (CDR) loop. Comparative structural studies have revealed that five of the six antigen-binding loops have only a limited repertoire of available three-dimensional structures. Each canonical structure can be characterized by the twist angle of the polypeptide backbone. Therefore, corresponding loops between antibodies can have very similar three-dimensional structures in most of the loops, despite high amino acid sequence variability (Chothia and Lesk, J.MoI. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J.MoI. Biol, 1996, 263:800). Furthermore, there is a correlation between the adopted loop structure and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the presence of amino acid residues in important positions within the loop and within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of these important amino acid residues.

[0154] The term "canonical structure" may also include considerations regarding the linear sequence of the antibody, as enumerated, for example, by Kabat (Kabat et al., cited above). Kabat's numbering scheme is a widely adopted standard for numbering amino acid residues of antibody variable domains in a consistent manner and is the preferred scheme applied in this invention, as also mentioned elsewhere in this specification. Further structural studies may be used to determine the canonical structure of the antibody. For example, differences not fully reflected by Kabat numbering can be described by the numbering scheme of Chothia et al. and / or revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence can be classified into a canonical class, among other things, for which appropriate chassis sequences can be identified (for example, based on the requirement to include various canonical structures in a library). The significance of Kabat numbering for antibody amino acid sequences, the structural studies described by Chothia et al. (cited above), and their implications for interpreting the canonical aspects of antibody structures are described in the literature. The subunit structures and three-dimensional arrangements of various classes of immunoglobulins are well known in this field. For an overview of antibody structures, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0155] Light chain CDR3, and especially heavy chain CDR3, can be the most important determinants of antigen binding within the light and heavy chain variable regions. In some antigen-binding molecules, heavy chain CDR3 appears to constitute the primary contact region between the antigen and the antibody. In vitro selection schemes that alter only the CDR3 can be used to change the binding properties of an antibody or to determine which residues contribute to antigen binding. Therefore, CDR3 is usually the greatest source of molecular diversity within the antibody binding site. For example, H3 can be as short as two amino acid residues or more than 26 amino acids.

[0156] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to the heavy (H) chain by a single covalent disulfide bond, whereas two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to the VH is usually referred to as CH1. The constant ("C") domain does not directly participate in antigen binding but exhibits various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation. The Fc region of the antibody is contained within the heavy chain constant domain and can interact with Fc receptors located, for example, on the cell surface.

[0157] There is a high degree of variability in the sequences of antibody genes after assembly and somatic mutation, and these variable genes are 10 10 It is presumed to encode two different antibody molecules (Immunoglobulin Genes, 2 nd (ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term “repertoire” refers to at least one nucleotide sequence that is derived in whole or in part from at least one sequence encoding at least one immunoglobulin. This sequence may be produced by in vivo rearrangement of the V, D, and J segments of the heavy chain and the V and J segments of the light chain. Alternatively, one or more sequences may be produced from cells in response to any rearrangement, for example, in vitro stimulation. Alternatively, some or all of this sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, for example, U.S. Patent No. 5,565,332). The repertoire may consist of only one sequence or may consist of multiple sequences, including those within a genetically diverse collection.

[0158] In relation to the present invention, the term "Fc moiety" or "Fc monomer" means a polypeptide comprising at least one domain having the function of the CH2 domain and at least one domain having the function of the CH3 domain of an immunoglobulin molecule. As is clear from the term "Fc monomer," polypeptides containing these CH domains are "polypeptide monomers." An Fc monomer may be a polypeptide comprising a fragment of the constant region of an immunoglobulin, excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but maintaining the functional portion of at least one CH2 domain and the functional portion of one CH3 domain, with the CH2 domain located on the amino-terminal side of the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a portion of the Ig-Fc hinge region, a CH2 region and a CH3 region, with the hinge region located on the amino-terminal side of the CH2 domain. The hinge region of the present invention is intended to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, by papain digestion of an immunoglobulin region (which naturally produces a dimer of two Fc polypeptides), but are not limited thereto. In another embodiment of this definition, the Fc monomer may be a polypeptide region containing parts of the CH2 and CH3 regions. Such Fc polypeptide molecules can be obtained, for example, by pepsin digestion of an immunoglobulin molecule, but are not limited thereto. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequences of the IgG1Fc, IgG2Fc, IgG3Fc, IgG4Fc, IgM Fc, IgA Fc, IgD Fc, and IgE Fc regions. (See, for example, Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because several variants exist among immunoglobulins, and simply for clarity, the Fc monomer refers to the two heavy chain constant-region immunoglobulin domains at the end of IgA, IgD, and IgG, and the three heavy chain constant-region immunoglobulin domains at the end of IgE and IgM. As mentioned, the Fc monomer may also include a movable hinge on the N-terminal side of these domains.In the case of IgA and IgM, the Fc monomer may contain a J chain. In the case of IgG, the Fc portion contains immunoglobulin domains CH2 and CH3, as well as a hinge between the first two domains and CH2. The boundaries of the Fc portion may differ, but an example of a human IgG heavy chain Fc portion containing the functional hinge, CH2 and CH3 domains may be defined, for example, as containing residues D231 (corresponding to D234 in Table 4 below) to P476 and L476 (in the case of IgG4), respectively, at the carboxyl terminology of the CH3 domain, according to Kabat numbering. Two Fc portions or Fc monomers fused to each other via a peptide linker define a third domain of the antigen-binding molecule of the present invention, which may also be defined as an scFc domain.

[0159] In some embodiments, the scFc domains disclosed herein (each consisting of fused Fc monomers) are assumed to be located only in the third domain of the antigen-binding molecule.

[0160] The IgG hinge region can be identified by similarity using Kabat numbering as shown in Table 4. In accordance with the above, the hinge domain / region is assumed to contain amino acid residues corresponding to the D234-P243 sequence of the IgG1 sequence by Kabat numbering. Similarly, the hinge domain / region is assumed to contain or consist of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 182) (corresponding to the D234-P243 sequence as shown in Table 4 below - variants of the sequence are also assumed, provided that the hinge region still promotes dimerization). In a preferred embodiment, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the antigen-binding molecule is removed by an N314X substitution (where X is any amino acid other than Q). The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat) V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0161] The third domain of the antigen-binding molecule may also contain or consist of DKTHTCPPCP(SEQ ID NO: 182)(i.e., hinge)-CH2-CH3-linker-DKTHTCPPCP(SEQ ID NO: 182)(i.e., hinge)-CH2-CH3 in the order of amino to carboxyl. In preferred embodiments, the peptide linker of the aforementioned antigen-binding molecule is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser(SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer of 5 or more (e.g., 5, 6, 7, 8, etc., or more), with 6 being preferred ((Gly4Ser)6). The construct may further contain the above-mentioned substitution N314X, preferably N314G, and / or further substitutions V321C and R309C. In preferred embodiments of the antigen-binding molecule as previously defined herein, the second domain is envisioned to bind to an extracellular epitope of the human and / or macaque CD3ε chain.

[0162] [Table 12]

[0163] In further embodiments, the hinge domain / region includes or comprises the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 183), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 184) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 185), and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 186). The IgG1 subtype hinge sequence may be the following sequence EPKSCDKTHTCPPCP (as shown in Table 4 and SEQ ID NO: 183). Accordingly, these core hinge regions are also conceivable in connection with the present invention.

[0164] The locations and sequences of the IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering described in Table 5.

[0165] [Table 13]

[0166] In one embodiment of the present invention, the amino acid residue highlighted in bold within the CH3 domain of the first or both Fc monomers is deleted.

[0167] The peptide linker into which the polypeptide monomers of the third domain ("Fc moieties" or "Fc monomers") are fused to each other preferably contains at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker contains at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferable that the linker contains up to 40 amino acid residues, more preferably up to 35 amino acid residues, and most preferably just 30 amino acid residues. A preferred embodiment of such a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187) or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer of 5 or more (e.g., 6, 7, or 8). Preferably, the integer is 6 or 7, and more preferably, the integer is 6.

[0168] When a linker is used to fuse a first domain with a second domain, or to fuse a first or second domain with a third domain, the linker is preferably of sufficient length and sequence to ensure that each of the first and second domains independently maintains their different binding specificities. With respect to peptide linkers that link at least two binding domains (or two variable domains) of the antigen-binding molecule of the present invention, it is preferable that such peptide linkers contain only a few amino acid residues (e.g., 12 amino acid residues or less). Accordingly, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Of the assumed peptide linkers having fewer than 5 amino acids, it is preferable that they contain 4, 3, 2, or 1 amino acid and are rich in Gly. A preferred embodiment of the peptide linker for fusion of the first and second domains is shown in SEQ ID NO: 1. A preferred linker embodiment of the peptide linker for fusion of the second and third domains is a (Gly)4-linker, which is a G4-linker.

[0169] In relation to one of the above-mentioned "peptide linkers," a particularly preferred "single" amino acid is Gly. Thus, the above-mentioned peptide linker may consist of a single amino acid Gly. In preferred embodiments of the present invention, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer of 1 or more (e.g., 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1 to 12. Features of the peptide linkers, including the absence of secondary structure enhancement, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Furthermore, a peptide linker that does not promote any secondary structure is preferred. The linking of the domains can be provided, for example, by genetic manipulation as described in the examples. Methods for preparing fused and operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., International Publication No. 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0170] In preferred embodiments of the antigen-binding molecule, the first and second domains form an antigen-binding molecule in a form selected from the group consisting of (scFv)2, scFv-single-domain mAb, diabody, and any oligomer of these forms.

[0171] In particular preferred embodiments, and as described in the appended examples, the first and second domains of the antigen-binding molecule of the present invention are a "bispecific single-chain antigen-binding molecule," more preferably a bispecific "single-chain Fv" (scFv). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked by a synthetic linker as described above herein, which allows them to be prepared using recombinant methods as a single protein chain paired so that the VL and VH regions form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). These antibody fragments can be obtained using prior art known to those skilled in the art, and the fragments can be evaluated for function in the same manner as complete or full-length antibodies. Therefore, a single-chain variable fragment (scFv) is a fusion protein of the variable region (VH) of the heavy chain and the variable region (VL) of the light chain of an immunoglobulin, typically linked by a short linker peptide of about 10 to 25 amino acids, preferably about 15 to 20 amino acids. The linker is usually rich in glycine for mobility and serine or threonine for solubility, and may link the N-terminus of the VH to the C-terminus of the VL or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0172] Bispecific single-chain antigen-binding molecules are known in the art and are described in International Publication No. 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loffler, Blood, (2000), 95, 6, 2098-2103, Bruhl, Immunol., (2001), 166, 2420-2426, and Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent No. 4,946,778, Kontermann and Dubel (2010) cited above, and Little (2009) cited above) can be adapted to generate single-chain antigen-binding molecules that specifically recognize a selected target.

[0173] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv having the form (scFv)2) can be produced by linking two scFv molecules (for example, using the linkers described above herein). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably called bivalent (i.e., it has a valency of 2 for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably called bispecific. This linking can be performed by generating a single peptide chain having two VH regions and two VL regions to obtain a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is the creation of scFv molecules containing a linker peptide that is too short (e.g., about 5 amino acids) to fold the two variable regions together, thus forcing the scFv to dimerize. This type is known as a diabody (see, for example, Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0174] The first, second, or first and second domains may each constitute a single-domain antibody, a variable domain of a single-domain antibody, or at least a CDR. A single-domain antibody contains only one (monomer) antibody variable domain that can selectively bind to a specific antigen independently of other V regions or domains. The first single-domain antibodies were produced from heavy-chain antibodies found in camels, and these are V H It is called an H fragment. Cartilaginous fish also have V NARIt possesses heavy chain antibodies (IgNARs) from which single-domain antibodies called fragments can be obtained. An alternative method is to split a dimeric variable domain derived from a common immunoglobulin of human or rodent origin into monomers, thereby obtaining a single-domain Ab as VH or VL. Most research on single-domain antibodies is currently based on heavy chain variable domains, but it has also been shown that light chain-derived nanobodies can specifically bind to target epitopes. Examples of single-domain antibodies are called sdAbs, nanobodies, or single variable-domain antibodies.

[0175] Therefore, (single-domain mAb)2 is V H , V L , V H H and V NAR It is a monoclonal antigen-binding molecule composed of at least two single-domain monoclonal antibodies individually selected from the group including the above. The linker is preferably in the form of a peptide linker. Similarly, "scFv-single-domain mAb" is a monoclonal antigen-binding molecule composed of at least one single-domain antibody as described above and one scFv molecule as described above. In this case as well, the linker is preferably in the form of a peptide linker.

[0176] Whether an antigen-binding molecule competes to bind to another given antigen-binding molecule can be measured using a competitive assay, such as a competitive ELISA or a cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Similar to avidin-coated ELISA plates, each of these beads can be used as a substrate when reacting with biotinylated proteins, and the assay can be performed on it. The antigen is coated onto the beads, then pre-coated with a first antibody. A secondary antibody is added to confirm any further binding. Flow cytometry is a possible method for reading the results.

[0177] T cells, or T lymphocytes, are a type of lymphocyte (a type of white blood cell) that plays a central role in cellular immunity. Several subsets of T cells exist, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a T cell receptor (TCR) on their cell surface. The TCR is involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules and is composed of two distinct protein chains. In 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain. When the TCR binds to antigen peptides and MHC (peptide / MHC complexes), the T lymphocyte is activated through a series of biochemical events mediated by related enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

[0178] The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains a CD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor-CD3 complex, which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are cell surface proteins of a highly related immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif, known as the immunoreceptor activation tyrosine motif, or ITAM, which is essential for TCR signaling. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on human chromosome 11. The most preferred epitopes of CD3 epsilon are contained within amino acid residues 1-27 of the human CD3 epsilon extracellular domain. The antigen-binding molecules according to the present invention are expected to exhibit less undesirable nonspecific T-cell activity in certain immunotherapies, which is typically and advantageously limited. In other words, this reduces the risk of side effects.

[0179] Lysis of redirected target cells via T cell recruitment by multispecific (or at least bispecific) antigen-binding molecules is accompanied by the formation of cytolytic synapses and the delivery of perforin and granzyme. The bound T cells are capable of sequential target cell lysis and are unaffected by immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, for example, International Publication No. 2007 / 042261).

[0180] The cytotoxicity mediated by the antigen-binding molecule of the present invention can be measured by various methods. Effector cells may be, for example, stimulated enriched (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin, or express or are transfected with the surface antigen of the macaque target cell bound by the first domain, the effector cells should also be of macaque origin, such as a macaque T cell line, e.g., 4119LnPx. The target cells should express the surface antigen of the target cell, e.g., the surface antigen of a human or macaque target cell (at least its extracellular domain). The target cells may be a cell line (e.g., CHO) that is stably or transiently transfected with the surface antigen of the target cell, e.g., the surface antigen of a human or macaque target cell. Alternatively, the target cells may be a native target cell line that expresses the surface antigen of the target cell. Typically, EC 50 The value is expected to be lower in target cell lines that express high levels of the target cell's surface antigen on the cell surface. The effector-to-target cell (E:T) ratio is typically about 10:1, but this can also vary. The cytotoxic activity of the target cell's surface antigen xCD3 bispecific antigen-binding molecule is, 51It can be measured in a Cr release assay (incubation time of approximately 18 hours) or a cytotoxicity assay using FACS (incubation time of approximately 48 hours). The incubation time (cytotoxicity reaction) of the assay can also be modified. Other methods for measuring cytotoxicity are known to those skilled in the art and include MTT or MTS assays, ATP system assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonality assays, and ECIS technology.

[0181] The cytotoxic activity mediated by the xCD3 bispecific antigen-binding molecule, which is the surface antigen of target cells in the present invention, is preferably measured by a cytotoxic assay using cells. 51 It may also be measured by a Cr release assay. Cytotoxic activity is EC 50 This is expressed as a value, which corresponds to the half-effect concentration (the concentration of the antigen-binding molecule that induces a cytotoxic response between the baseline and the maximum value). Preferably, the EC of the xCD3 bispecific antigen-binding molecule, which is the surface antigen of the target cell. 50 The values ​​are ≤5000pM or ≤4000pM, more preferably ≤3000pM or ≤2000pM, even more preferably ≤1000pM or ≤500pM, even more preferably ≤400pM or ≤300pM, even more preferably ≤200pM, even more preferably ≤100pM, even more preferably ≤50pM, even more preferably ≤20pM or ≤10pM, and most preferably ≤5pM.

[0182] The above given EC 50 The value can be measured using various assays. Stimulated / enriched CD8 + When T cells are used as effector cells, EC is superior to unstimulated PBMCs. 50 Those skilled in the art are aware that the value can be expected to decrease. Furthermore, EC 50 The values ​​can be expected to be lower compared to rats with less target expression when target cells express a large number of target cell surface antigens. For example, stimulated / enriched human CD8 +When using T cells as effector cells (and when using cells transfected with the surface antigen of target cells such as CHO cells or human cell lines positive for the surface antigen of target cells as target cells), the EC of the xCD3 bispecific antigen-binding molecule of the target cell's surface antigen 50 The value is preferably ≤1000pM, more preferably ≤500pM, even more preferably ≤250pM, even more preferably ≤100pM, even more preferably ≤50pM, even more preferably ≤10pM, and most preferably ≤5pM. When human PBMCs are used as effector cells, the EC of the target cell surface antigen × CD3 bispecific antigen-binding molecule 50 The value is preferably ≤5000pM or ≤4000pM (especially when the target cells are human cell lines positive for the surface antigen of the target cells), more preferably ≤2000pM (especially when the target cells are cells transfected with the surface antigen of the target cells, such as CHO cells), more preferably ≤1000pM or ≤500pM, even more preferably ≤200pM, even more preferably ≤150pM, even more preferably ≤100pM, and most preferably ≤50pM. When using a macaque T cell line such as LnPx4119 as an effector cell and a cell line transfected with the surface antigen of macaque target cells such as CHO cells as the target cell line, the EC of the xCD3 bispecific antigen-binding molecule of the target cell surface antigen is used. 50 The value is preferably ≤2000pM or ≤1500pM, more preferably ≤1000pM or ≤500pM, even more preferably ≤300pM or ≤250pM, even more preferably ≤100pM, and most preferably ≤50pM.

[0183] Preferably, the target cell surface antigen × CD3 bispecific antigen-binding molecule of the present invention does not induce or mediate the lysis of target cell surface antigen-negative cells, such as CHO cells, or substantially induces or mediates the lysis. The terms “does not induce lysis,” “substantially does not induce lysis,” “does not mediate lysis,” or “substantially does not mediate lysis” mean that, with the lysis of target cell surface antigen-positive human cell lines being 100%, the antigen-binding molecule of the present invention does not induce or mediate the lysis of target cell surface antigen-negative cells by more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5%. This typically applies to antigen-binding molecule concentrations of up to 500 nM. Those skilled in the art know how to measure cell lysis without further effort. Furthermore, specific instructions for methods of measuring cell lysis are taught herein.

[0184] The difference in cytotoxic activity between the monomeric and dimeric isoforms of the xCD3 bispecific antigen-binding molecule, which is a surface antigen of individual target cells, is called the "potency gap." This potency gap is, for example, the EC of the monomeric form of the molecule. 50 Value and dimeric form of EC 50 It can be calculated as a ratio between the values. The efficacy gap of the xCD3 bispecific antigen-binding molecule for the target cell surface antigen of the present invention is preferably ≤5, more preferably ≤4, even more preferably ≤3, even more preferably ≤2, and most preferably ≤1.

[0185] The first and / or second (or any further) binding domains of the antigen-binding molecule of the present invention are preferably interspecies-specific to members of the order Mammalia of primates. Interspecies-specific CD3-binding domains are described, for example, in International Publication No. 2008 / 119567. According to one embodiment, the first and / or second binding domains bind to the surface antigens / CD3 of target cells of primates, including (but not limited to) New World primates (such as Callisrix jacchus, Saguinus Oedipus, or Saimiri sciureus), Old World primates (such as baboons and macaques), gibbons, and non-human homininaes, respectively, in addition to binding to the surface antigens and human CD3 of human target cells.

[0186] In one embodiment of the antigen-binding molecule of the present invention, the first domain binds to the surface antigen of human target cells and further binds to the surface antigen of macaque target cells, such as the surface antigen of Macaca fascicularis target cells, more preferably to the surface antigen of macaque target cells expressed on the surface of macaque cells. The affinity of the first binding domain for the surface antigen of macaque target cells is preferably ≤15 nM, more preferably ≤10 nM, even more preferably ≤5 nM, even more preferably ≤1 nM, even more preferably ≤0.5 nM, even more preferably ≤0.1 nM, most preferably ≤0.05 nM, or even more preferably ≤0.01 nM.

[0187] Preferably, the binding affinity gap of the antigen-binding molecule according to the present invention between the surface antigen of macaque target cells and the surface antigen of human target cells [ma target cell surface antigen: hu target cell surface antigen] (determined, for example, by BiaCore or scatchard analysis) is <100, preferably <20, more preferably <15, even more preferably <10, even more preferably <8, even more preferably <6, and most preferably <2. The preferred range for the binding affinity gap of the antigen-binding molecule according to the present invention between the surface antigen of macaque target cells and the surface antigen of human target cells is 0.1 to 20, more preferably 0.2 to 10, even more preferably 0.3 to 6, even more preferably 0.5 to 3 or 0.5 to 2.5, and most preferably 0.5 to 2 or 0.6 to 2.

[0188] The second (binding) domain of the antigen-binding molecule binds to human CD3 epsilon and / or macaque CD3 epsilon. In preferred embodiments, the second domain further binds to Callisrix jacchus, Saguinus oedipus, or Saimiri sciureus CD3 epsilon. Both Callisrix jacchus and Saguinus oedipus are New World primates belonging to the family Callitrichidae, while Saimiri sciureus is a New World primate belonging to the family Cebidae.

[0189] For this antigen-binding molecule, the second binding domain that binds to the extracellular epitope of human and / or macaque CD3 preferably includes a VL region containing CDR-L1, CDR-L2, and CDR-L3 selected from the following: (a) CDR-L1 as shown in Sequence ID 27 of International Publication No. 2008 / 119567, CDR-L2 as shown in Sequence ID 28 of International Publication No. 2008 / 119567, and CDR-L3 as shown in Sequence ID 29 of International Publication No. 2008 / 119567 (b) CDR-L1 as indicated by Sequence ID 117 of International Publication No. 2008 / 119567, CDR-L2 as indicated by Sequence ID 118 of International Publication No. 2008 / 119567, and CDR-L3 as indicated by Sequence ID 119 of International Publication No. 2008 / 119567, and (c) CDR-L1 as indicated by Sequence ID 153 of International Publication No. 2008 / 119567, CDR-L2 as indicated by Sequence ID 154 of International Publication No. 2008 / 119567, and CDR-L3 as indicated by Sequence ID 155 of International Publication No. 2008 / 119567.

[0190] In a more preferred embodiment of this antigen-binding molecule, the second domain that binds to the extracellular epitope of the human and / or macaque CD3 epsilon chain comprises a VH region including CDR-H1, CDR-H2, and CDR-H3 selected from the following: (a) CDR-H1 as shown in Sequence ID 12 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 13 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 14 of International Publication No. 2008 / 119567; (b) CDR-H1 as indicated by Sequence ID 30 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 31 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 32 in International Publication No. 2008 / 119567; I. CDR-H1 as indicated by Sequence ID 48 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 49 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 50 in International Publication No. 2008 / 119567; (d) CDR-H1 as indicated by Sequence ID 66 of International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 67 of International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 68 of International Publication No. 2008 / 119567; I. CDR-H1 as indicated by Sequence ID 84 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 85 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 86 in International Publication No. 2008 / 119567; (f) CDR-H1 as indicated by Sequence ID 102 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 103 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 104 in International Publication No. 2008 / 119567; (g) CDR-H1 as indicated by Sequence ID 120 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 121 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 122 in International Publication No. 2008 / 119567; (h) CDR-H1 as indicated by Sequence ID 138 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 139 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 140 in International Publication No. 2008 / 119567; (i) CDR-H1 as indicated by Sequence ID 156 of International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 157 of International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 158 of International Publication No. 2008 / 119567, (j) CDR-H1 as indicated by Sequence ID 174 in International Publication No. 2008 / 119567, CDR-H2 as indicated by Sequence ID 175 in International Publication No. 2008 / 119567, and CDR-H3 as indicated by Sequence ID 176 in International Publication No. 2008 / 119567.

[0191] In a preferred embodiment of the antigen-binding molecule, the three groups of VL CDRs described above are combined with the ten groups of VH CDRs described above in the second binding domain to form a (30) group comprising CDR-L1~3 and CDR-H1~3, respectively.

[0192] With respect to the antigen-binding molecule, it is preferable that the second domain that binds to CD3 includes a VL region selected from the group consisting of VL regions such as those shown in SEQ ID NOs. 25, 346, or 354; or those shown in SEQ ID NOs. 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179, or 183 of International Publication No. 200 / 119567; or SEQ ID NO. 200.

[0193] It is also preferable that the second domain bound to CD3 includes a VH region selected from the group consisting of VH regions such as those shown in Sequence ID No. 25, 346, or 354; or those shown in Sequence ID No. 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177, or 181 of International Publication No. 2008 / 119567, or those shown in Sequence ID No. 201.

[0194] More preferably, the antigen-binding molecule is characterized by a second domain that binds to CD3ε, including a VL region and a VH region selected from the group consisting of the following: (a) the VL area shown in Sequence ID No. 17 or 21 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID No. 15 or 19 of International Publication No. 2008 / 119567; (b) The VL area shown in Sequence ID 35 or 39 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 33 or 37 of International Publication No. 2008 / 119567; I. VL areas as indicated by Sequence ID 53 or 57 in International Publication No. 2008 / 119567 and VH areas as indicated by Sequence ID 51 or 55 in International Publication No. 2008 / 119567; (d) The VL area shown in Sequence ID 71 or 75 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 69 or 73 of International Publication No. 2008 / 119567; I. Very Large (VL) areas as indicated by Sequence ID 89 or 93 in International Publication No. 2008 / 119567 and Very Large (VH) areas as indicated by Sequence ID 87 or 91 in International Publication No. 2008 / 119567; (f) The VL area shown in Sequence ID 107 or 111 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 105 or 109 of International Publication No. 2008 / 119567; (g) the VL area shown in Sequence ID 125 or 129 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 123 or 127 of International Publication No. 2008 / 119567; (h) The VL area as shown in Sequence ID 143 or 147 of International Publication No. 2008 / 119567 and the VH area as shown in Sequence ID 141 or 145 of International Publication No. 2008 / 119567; (i) the VL area shown in Sequence ID No. 161 or 165 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID No. 159 or 163 of International Publication No. 2008 / 119567; and (j) The VL area shown in Sequence ID 179 or 183 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 177 or 181 of International Publication No. 2008 / 119567.

[0195] A second domain that binds to CD3, including a VL region as shown in SEQ ID NO: 200 and a VH region as shown in SEQ ID NO: 201, is also preferred in relation to the antigen-binding molecule.

[0196] According to a preferred embodiment of the antigen-binding molecule, the first and / or second domains have the following configuration: the pair of VH and VL regions is in the form of a single-chain antibody (scFv). The VH and VL regions are arranged in the order VH-VL or VL-VH. Preferably, the VH region is located at the N-terminus of the linker sequence and the VL region is located at the C-terminus of the linker sequence.

[0197] A preferred embodiment of the TCE described above is characterized by a domain that binds to CD3ε, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187 of International Publication No. 2008 / 119567, or a sequence having 90, 95, 99, or 100% sequence identity with SEQ ID NOs: 26, 381, 382, ​​or 383.

[0198] Covalent modification of antigen-binding molecules is also included within the scope of the present invention, and this is generally performed post-translation, although not necessarily. For example, some types of covalent modification of antigen-binding molecules are introduced into the molecule by reacting specific amino acid residues of the antigen-binding molecule with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0199] Cysteinyl residues most commonly react with α-haloacetates (and their corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidemethyl derivatives. Cysteinyl residues can also be derivatized by reactions with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercrinebenzoic acid, 2-chloromercrine-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0200] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific to the histidyl side chain. Para-bromophenacyl bromide is also useful, and this reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Ricinyl and amino-terminal residues react with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the ricinyl residue. Other suitable reagents for derivatization of alpha-amino-containing residues include imide esters such as methyl picolinimide; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylates.

[0201] Arginine residues are modified by reaction with one or more conventional reagents, particularly phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires the reaction to be carried out under alkaline conditions. Furthermore, these reagents can react with lysine groups and arginine epsilon-amino groups.

[0202] Specific modification of tyrosyl residues may be performed, particularly to introduce spectral labeling to tyrosyl residues through reactions with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidisole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 The chloramine T method described above, which involves iodizing tyrosyl residues using I to prepare a labeled protein for use in radioimmunoassays, is preferred.

[0203] The carboxyl side group (aspartyl or glutamyl) is selectively modified by reaction with a carbodiimide (R'-N=C=N--R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, the aspartyl and glutamyl residues are converted to asparaginyl and glutamyl residues by reaction with ammonium ions.

[0204] Derivatization with difunctional substances is useful for crosslinking antigen-binding molecules to water-insoluble support matrices or surfaces for use in various ways. Commonly used crosslinking agents include, for example, N-hydroxysuccinimide esters such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and esters with 4-azidosalicylic acid, homodifunctional imide esters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and difunctional maleimides such as bis-N-maleimide-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive non-water-soluble matrices such as cyanide-activated carbohydrates, and reactive substrates as described in U.S. Patents No. 3,969,287; No. 3,691,016; No. 4,195,128; No. 4,247,642; No. 4,229,537; and No. 4,330,440, can be used for protein immobilization.

[0205] Glutaminyl and asparaginyl residues are frequently deamidated to their corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under weakly acidic conditions. Any form of these residues is within the scope of the present invention.

[0206] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0207] Another type of covalent modification of antigen-binding molecules that falls within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, the glycosylation pattern can depend on both the protein sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as discussed below) or on the host cell or organism in which the protein is produced. Detailed expression systems are discussed below.

[0208] Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the binding of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic binding of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide generates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine (although 5-hydroxyproline or 5-hydroxylysine may also be used).

[0209] The addition of a glycosylation site to an antigen-binding molecule is conveniently achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above (in the case of an N-linked glycosylation site). This modification can be carried out by adding or substituting one or more serine or threonine residues to the start sequence (in the case of an O-linked glycosylation site). In short, it is preferable to modify the amino acid sequence of the antigen-binding molecule by changing it at the DNA level, particularly by mutating the polypeptide-encoding DNA with pre-selected bases to generate codons that translate to the desired amino acids.

[0210] Another means of increasing the number of sugar chains on an antigen-binding molecule is by chemically or enzymatically attaching glycosides to the protein. These procedures are advantageous in that they do not require the production of the protein in a host cell with glycosylation ability for N-linked and O-linked glycosylation. Depending on the mode of attachment used, sugars can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group such as that of cysteine, (d) a free hydroxyl group such as that of serine, threonine, or hydroxyproline, (e) an aromatic residue such as that of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine. These methods are described in International Publication No. 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0211] The removal of sugar chains present on the starting antigen-binding molecule can be performed chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves almost all or all of the sugars except for the bound sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of the carbohydrate portion on the polypeptide can be achieved by using various endo- and exo-glycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin inhibits the formation of protein-N-glycosidic bonds.

[0212] Other modifications of antigen-binding molecules are also considered herein. For example, another type of covalent modification of antigen-binding molecules involves linking the antigen-binding molecule to various non-proteinoid polymers, which include, but are not limited to, various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antigen-binding molecule to facilitate the addition of polymers such as PEG.

[0213] In some embodiments, the covalent modification of the antigen-binding molecule of the present invention involves the addition of one or more labels. The labeling groups may be attached to the antigen-binding molecule via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used when carrying out the present invention. The terms “label” or “labeling group” refer to any detectable label. Generally, labels are divided into various classes depending on the assay in which they are detected, and include, but are not limited to, the following examples. a) Radioactive isotopes or radionuclides (for example, 3 H, 14 C, 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 I) Isotopic labels that may be radioactive isotopes or heavy isotopes b) Magnetic labeling (e.g., magnetic particles) c) Redox-active moiety d) Optical dyes (including, but not limited to, chromophores, phosphors and fluorophores), such as fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups and fluorophores which may be either "low molecular weight" phosphors or protein phosphors. e) Enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) f) Biotinylation group g) A predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal-binding domain, an epitope tag, etc.).

[0214] "Fluorescent label" refers to any molecule that can be detected by its intrinsic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes (Eugene, OR), FITC, Rhodamine and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes containing fluorophores are listed in Richard P. Haugland's Molecular Probes Handbook.

[0215] Suitable protein-based fluorescent labels also include green fluorescent protein (GFP) from Renilla, Ptilosarcus, and Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), and blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8 th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), highly sensitive yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renilla (International Publication No. 92 / 15673, International Publication No. 95 / 07463, International Publication No. 98 / 14605, International Publication No. 98 / 26277, International Publication No. 99 / 49019) Other examples include, but are not limited to, U.S. Patent Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558.

[0216] The antigen-binding molecules of the present invention may also include additional domains that are useful, for example, for isolating the molecule or relating to the molecule's adapted pharmacokinetic profile. Domains useful for isolating the antigen-binding molecule may be selected from peptide motifs that can be captured by isolation methods, such as isolation columns, or auxiliaryly introduced portions. Non-limiting embodiments of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and their variants (e.g., StrepII tags), and His tags. All antigen-binding molecules disclosed herein, characterized by identified CDRs, may include His-tagged domains, commonly known as repeats of consecutive His residues, preferably five, more preferably six His residues (hexahistidine), in the amino acid sequence of the molecule. The His tag may be located, for example, at the N-terminus or C-terminus of the antigen-binding molecule, preferably at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 199) is ligated to the C-terminus of the antigen-binding molecule according to the present invention via a peptide bond. In addition, a PLGA-PEG-PLGA conjugate system may be combined with the polyhistidine tag to improve sustained release and pharmacokinetic profile.

[0217] Amino acid sequence modifications of the antigen-binding molecules described herein are also intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecules. Variants of the amino acid sequence of the antigen-binding molecules are prepared by introducing appropriate nucleotide changes into the nucleic acid of the antigen-binding molecule or by synthesizing peptides. All of the amino acid sequence modifications described below should result in antigen-binding molecules that continue to retain the desired biological activity of the unmodified parent molecule (binding to surface antigens and CD3 on target cells).

[0218] The term "amino acid" or "amino acid residue" typically refers to an amino acid with a definition recognized in the art, such as an amino acid selected from the group consisting of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V), but modified amino acids, synthetic amino acids, or rare amino acids may be used as needed. Generally, amino acids can be classified by the presence of nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Giu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gin, Giy, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0219] Amino acid modifications include, for example, deletions of residues from and / or insertions of residues and / or substitutions of residues within the amino acid sequence of an antigen-binding molecule. Any combination of deletions, insertions, and substitutions is performed to arrive at the final construct, provided that the final construct has the desired properties. Furthermore, by altering the amino acids, it is also possible to change the post-translational processes of the antigen-binding molecule, such as changing the number or position of glycosylation sites.

[0220] For example, one, two, three, four, five, or six amino acids may be inserted, substituted, or deleted in each of the CDRs (naturally, depending on their length), while one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, nine, ten, one, two, three, three, four, nine, one

[0221] The most important sites for substitutional mutagenesis are the CDRs of the heavy chain and / or light chain, particularly the hypervariable region (HDR), but modifications of the FRs in the heavy chain and / or light chain are also intended. Substitutions are preferably conservative substitutions as described herein. Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework region (FR). For example, if the CDR sequence contains 6 amino acids, 1, 2, or 3 of these amino acids are expected to be substituted. Similarly, if the CDR sequence contains 15 amino acids, 1, 2, 3, 4, 5, or 6 of these amino acids are expected to be substituted.

[0222] A useful method for identifying specific residues or regions of antigen-binding molecules that are favorably positioned for mutagenesis is called the "alanine scanning mutagenesis method," as described by Cunningham and Wells in Science, 244:1081-1085 (1989). This method involves identifying residues or target residue groups within the antigen-binding molecule (e.g., charged residues such as arg, asp, his, lys, and glu) and replacing them with neutral or negatively charged amino acids (most preferably alanine or polyalanine) that influence the interaction between the amino acid and the epitope.

[0223] Next, by introducing further or other variants at the substitution site, i.e., in place of the substitution site, the range of amino acid positions that are functionally sensitive to the substitution is carefully selected. Thus, although the site or region to introduce the amino acid sequence mutation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze or optimize the performance of the mutation at a given site, alanine scanning or random mutagenesis may be performed at the target codon or target region to clean whether the expressed antigen-binding molecule variant is the optimal combination of desired activity. Techniques for introducing substitution mutations at predetermined sites in DNA with known sequences are well known, and include, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of variants is performed using antigen-binding activity assays such as target cell surface antigen or CD3 binding.

[0224] Generally, when one or more or all of the CDRs of the heavy chain and / or light chain are substituted with amino acids, the resulting “substituted” sequence is preferably at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the “original” CDR sequence. This means that the degree of identity with the “substituted” sequence depends on the length of the CDR. For example, for a CDR with five amino acids to have at least one substituted amino acid, it is preferable that it be 80% identical to the substituted sequence. Therefore, the CDRs of antigen-binding molecules may have varying degrees of identity with respect to their substituted sequences; for example, CDRL1 may have 80% identity, while CDRL3 may have 90% identity.

[0225] A preferred substitution (or replacement) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more of the “exemplary substitutions” listed in Table 3 below) is considered, as long as the antigen-binding molecule retains its ability to bind to the surface antigen of the target cell via the first domain and to CD3 or CD3 epsilon via the second domain, and / or its CDR is identical to the substituted sequence (at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the “original” CDR sequence).

[0226] Conservative substitutions are shown under the heading “Preferred Substitutions” in Table 3. If such substitutions alter biological activity, they are referred to as “Exemplary Substitutions” in Table 3, or substantial changes may be introduced, as further described below by referring to classes of amino acids, and the product may be screened for desired characteristics.

[0227] [Table 14]

[0228] Substantial modification of the biological properties of antigen-binding molecules is achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone of the substitution region, for example, as a sheet-like or helical three-dimensional structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulkiness of the side chain. Naturally occurring residues are classified into the following groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophobic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0229] Non-conservative substitutions involve exchanging one member of one class for another. To avoid abnormal crosslinking, any cysteine ​​residue that does not contribute to maintaining the proper three-dimensional structure of the antigen-binding molecule can be substituted, generally with serine, to improve the oxidative stability of the molecule. Conversely, the stability of an antibody can be improved by adding a cysteine ​​bond (especially if the antibody is an antibody fragment such as an Fv fragment).

[0230] With respect to amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, such as, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444; computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis); the Best Fit sequence program, preferably with default settings, as described by Develeux et al., 1984, Nucl. Acid Res. 12:387-395; or by visual inspection. Preferably, the identity percentage is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and join penalty of 30, “Current Methods in Sequence Comparison and Analysis”, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0231] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create multiple sequence alignments from a group of related sequences. This also allows plotting a tree showing the clustering relationships used to generate the alignments. PILEUP uses a simplified version of the progressive alignment method described by Feng & Doolittle, 1987, J.Mol.Evol.35:351-360; this method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap.

[0232] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J.Mol.Biol.215:403-410; Altschul et al., 1997, Nucleic Acids Res.25:3389-3402; and Karin et al., 1993, Proc.Natl.Acad.Sci.USA90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to their default values. The adjustable parameters are set to the following values: overlap span=1, overlap fraction=0.125, word threshold(T)=II. The HSP S-parameter and HSP S2-parameter are dynamic values, constructed by the program itself depending on the composition of a particular sequence and the composition of a particular database from which the target sequence is searched, but their values ​​can be adjusted to increase sensitivity.

[0233] A further useful algorithm is gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores, with the threshold T parameter set to 9, and a two-hit method resulting in gapless extension with a cost of 10+k for a gap length k, where Xu is set to 16 and Xg is set to 40 during the database search phase and 67 during the algorithm's output phase. Gapped alignment is initiated with a score corresponding to approximately 22 bits.

[0234] Generally, the amino acid homology, similarity, or identity between individual variant CDR or VH / VL sequences is at least 60% with respect to the sequences shown herein, and more typically, the homology or identity increases to at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and nearly 100%. Similarly, the “percentage of nucleic acid sequence identity (%)” for the nucleic acid sequences of binding proteins identified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to nucleotide residues in the coding sequence of the antigen-binding molecule. In a specific method, the BLASTN module of WU-BLAST-2 is used with default parameters set to overlap span and overlap fraction of 1 and 0.125, respectively.

[0235] Generally, the nucleic acid sequence homology, similarity, or identity between the nucleotide sequences encoding individual variant CDR or VH / VL sequences and the nucleotide sequences shown herein is at least 60%, and more typically, it is preferable that the homology or identity increases to at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and nearly 100%. Accordingly, the "variant CDR" or "variant VH / VL region" has specific homology, similarity or identity with respect to the parent CDR / VH / VL of the present invention and shares a biological function that includes, but is not limited to, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0236] In one embodiment, the percentage of identity of the antigen-binding molecule according to the present invention to human germline is ≥70% or ≥75%, more preferably ≥80% or ≥85%, even more preferably ≥90%, most preferably ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, or even further ≥96%. Identity to human antibody germline gene products is considered an important feature for reducing the risk that therapeutic proteins will induce an immune response to a drug in a patient undergoing treatment. Hwang & Foote ("Immunogenicity of engineered Antibodies"; Methods 36(2005)3-10) have demonstrated that reducing the non-human portion of a drug-antigen-binding molecule reduces the risk of inducing anti-drug antibodies in a patient undergoing treatment. By comparing a vast number of clinically evaluated antibody drugs and their respective immunogenicity data, humanization of the V region of antibodies tends to result in lower protein immunogenicity (average 5.1% of patients) compared to antibodies possessing the unmodified, non-human V region (average 23.59% of patients). Therefore, for V region-based protein therapeutics in the form of antigen-binding molecules, a high degree of identity with the human sequence is desirable. To determine this germline identity, the V region of VL can be aligned with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the amino acid sequence can be calculated as a percentage by dividing the number of identical amino acid residues by the total number of amino acid residues in VL. A similar method is possible for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), however, VH CDR3 may be excluded due to its high diversity and the lack of existing alignment partners for human germline VH CDR3. Next, recombinant technology can be used to increase sequence identity for human antibody germline genes.

[0237] In further embodiments, the bispecific antigen-binding molecule exhibits high monomer yield under standard research-scale conditions, for example, in a standard two-step purification process. Preferably, the monomer yield of the antigen-binding molecule according to the present invention is ≥0.25 mg per L of supernatant, more preferably ≥0.5 mg per L, even more preferably ≥1 mg per L, and most preferably ≥3 mg per L of supernatant.

[0238] Similarly, the yield of isoforms of the dimeric antigen-binding molecule, and therefore the percentage of monomers of the antigen-binding molecule (i.e., monomer:(monomer+dimer)), can be determined. The productivity of monomeric and dimeric antigen-binding molecules, as well as the calculated monomer percentages, can be obtained, for example, in the SEC purification step of the culture supernatant from production on a standardized study scale in a roller bottle. In one embodiment, the monomer percentage of the antigen-binding molecule is ≥80%, more preferably ≥85%, even more preferably ≥90%, and most preferably ≥95%.

[0239] In one embodiment, the antigen-binding molecule preferably has plasma stability (ratio of EC50 in the presence of plasma to EC50 in the absence of plasma) of ≤5 or ≤4, more preferably ≤3.5 or ≤3, even more preferably ≤2.5 or ≤2, and most preferably ≤1.5 or ≤1. The plasma stability of the antigen-binding molecule is determined by incubating the construct in human plasma at 37°C for 24 hours, followed by 51This can be verified by determining the EC50 using a chromium-releasing cytotoxicity assay. Effector cells in the cytotoxicity assay may be stimulated, enriched human CD8-positive T cells. Target cells may be, for example, CHO cells transfected with the surface antigen of human target cells. An effector cell to target cell (E:T) ratio of 10:1 can be selected. The human plasma pool used for this purpose is derived from the blood of healthy donors collected using EDTA-coated syringes. Cellular components are removed by centrifugation, the upper plasma phase is collected, and then pooled. As a control, antigen-binding molecules are diluted in RPMI-1640 medium immediately before the cytotoxicity assay. Plasma stability is calculated as the ratio of EC50 (after plasma incubation) to EC50 (control).

[0240] It is even more preferable that the monomer-to-dimer conversion rate of the antigen-binding molecule of the present invention be low. The conversion rate can be measured under different conditions and analyzed by high-speed size exclusion chromatography. For example, incubation of the monomer isoform of the antigen-binding molecule can be carried out in an incubator at 37°C for 7 days at a concentration of, for example, 100 μg / ml or 250 μg / ml. Under these conditions, it is preferable that the percentage of dimers of the antigen-binding molecule of the present invention is ≤5%, more preferably ≤4%, even more preferably ≤3%, even more preferably ≤2.5%, even more preferably ≤2%, even more preferably ≤1.5%, most preferably ≤1%, or ≤0.5%, or even further 0%.

[0241] Furthermore, it is preferable that the bispecific antigen-binding molecule exhibits a very low dimerization rate after several freeze / thaw cycles. For example, the antigen-binding molecule monomer is adjusted to a concentration of 250 μg / ml in, for example, a general formulation buffer, subjected to three freeze / thaw cycles (freezing at -80°C for 30 minutes, followed by thawing at room temperature for 30 minutes), and then a high-speed SEC is performed to determine the percentage of the initial monomer antigen-binding molecule converted to a dimer antigen-binding molecule. Preferably, the percentage of dimers of the bispecific antigen-binding molecule is, for example, ≤5%, more preferably ≤4%, even more preferably ≤3%, even more preferably ≤2.5%, even more preferably ≤2%, even more preferably ≤1.5%, and most preferably ≤1% or even ≤0.5% after three freeze / thaw cycles.

[0242] Bispecific antigen-binding molecules preferably exhibit good thermal stability with an aggregation temperature of ≥45°C or ≥50°C, more preferably ≥52°C or ≥54°C, even more preferably ≥56°C or ≥57°C, and most preferably ≥58°C or ≥59°C. From the viewpoint of antibody aggregation temperature, the thermal stability parameter can be determined as follows: A 250 μg / mL antibody solution is transferred to a single-use cuvette and placed in a dynamic light scattering (DLS) instrument. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min while continuously acquiring the measured radius. An increase in radius indicates protein melting and aggregation, which is used to calculate the antibody aggregation temperature.

[0243] Alternatively, the melting temperature curve can be measured by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of the antigen-binding molecule. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. Energy uptake of the sample containing the antigen-binding molecule is recorded from 20°C to 90°C and compared with a sample containing only the formulation buffer. The antigen-binding molecule is adjusted to a final concentration of 250 μg / ml in, for example, SEC running buffer. The temperature of the entire sample is gradually increased to record each melting curve. Energy uptake of the sample and the formulation buffer standard is recorded at each temperature T. The difference in energy uptake Cp (kcal / mole / °C) obtained by subtracting the standard from the sample is plotted against each temperature. The melting temperature is defined as the temperature at the first maximum value of energy uptake.

[0244] The xCD3 bispecific antigen-binding molecule of the target cell surface antigen of the present invention is also assumed to have a turbidity of ≤0.2, preferably ≤0.15, more preferably ≤0.12, even more preferably ≤0.1, and most preferably ≤0.08 (measured by OD340 after concentrating the purified monomer antigen-binding molecule to 2.5 mg / ml and incubating overnight).

[0245] It is further anticipated that bispecific antigen-binding molecules may exhibit therapeutic efficacy or antitumor activity. This can be evaluated, for example, in the tests disclosed in the following examples of advanced-stage human tumor xenograft models.

[0246] Those skilled in the art know how to obtain meaningful and reproducible results while modifying or adapting specific parameters of the present test, such as the number of tumor cells injected, the injection site, the number of human T cells transplanted, the amount of bispecific antigen-binding molecule administered, and the schedule. Preferably, the tumor growth inhibitory T / C [%] is ≤70 or ≤60, more preferably ≤50 or ≤40, even more preferably ≤30 or ≤20, most preferably ≤10 or ≤5, or even further ≤2.5.

[0247] In a preferred embodiment of the antigen-binding molecule, the antigen-binding molecule is a single-chain antigen-binding molecule.

[0248] Furthermore, in a preferred embodiment of the antigen-binding molecule, the third domain is arranged in the order of amino to carboxyl. Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 This also includes.

[0249] In one embodiment of the present invention, the CH2 domain of one or preferably each (both) of the third domains of the polypeptide monomer also includes an intradomain cysteine ​​disulfide crosslink. As is well known in the art, the term "cysteine ​​disulfide crosslink" refers to a functional group having the general structure RSSR. This linkage is also called an SS bond or disulfide crosslink and is obtained by the coupling of two thiol groups of a cysteine ​​residue. With respect to the antigen-binding molecule of the present invention, it is particularly preferable that the cysteine ​​forming the cysteine ​​disulfide crosslink in the mature antigen-binding molecule be introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0250] In one embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of the glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid other than Q. The above substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321).

[0251] For example, a preferred feature of the antigen-binding molecule of the present invention compared to a bispecific hetero-Fc antigen-binding molecule known in the art (Figure 1b) may be related, in particular, to the introduction of the above-mentioned modification in the CH2 domain. Accordingly, with respect to the construct of the present invention, it is preferable that the CH2 domain in the third domain of the antigen-binding molecule of the present invention contains intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321, and / or that the glycosylation site at Kabat position 314 is removed by N314X substitution, preferably N314G substitution, as described above.

[0252] In a more preferred embodiment of the present invention, the CH2 domain in the third domain of the antigen-binding molecule of the present invention includes intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 is removed by N314G substitution.

[0253] In one embodiment, the present invention is an antigen-binding molecule, (182) The first domain contains two antibody-variable domains, and the second domain contains two antibody-variable domains; (ii) The first domain contains one antibody variable domain, and the second domain contains two antibody variable domains; (iii) The first domain contains two antibody variable domains and the second domain contains one antibody variable domain; or (iv) The first domain contains one antibody variable domain, and the second domain contains one antibody variable domain, We provide antibody constructs.

[0254] Accordingly, the first and second domains may be binding domains containing two antibody-variable domains, such as a VH domain and a VL domain. Examples of such binding domains containing two antibody-variable domains as described above in this specification include, for example, the Fv fragment, scFv fragment, or Fab fragment as described above in this specification. Alternatively, one or both of these binding domains may contain only a single variable domain. Examples of such single-domain binding domains as described above in this specification include, for example, nanobody or single-variable-domain antibodies containing only one variable domain, which may be VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0255] In preferred embodiments of the antigen-binding molecule of the present invention, the first and second domains are fused to a third domain via a peptide linker. Preferred peptide linkers are described above herein and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187) or a polymer thereof, i.e., (Gly4Ser)x (where x is an integer of 1 or more (e.g., 2 or 3)). A particularly preferred linker for the fusion of the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0256] In a preferred embodiment, the antigen-binding molecule of the present invention is composed of amino and carboxyl molecules in that order. (a) The first domain; (b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 187-189; I. Second domain; (d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; I. The first polypeptide monomer of the third domain; (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) Second polypeptide monomer of the third domain It is characterized by including.

[0257] In one aspect of the present invention, the surface antigen of a target cell bound by the first domain is a tumor antigen, an antigen specific to an immunodeficiency, or a viral antigen. As used herein, the term “tumor antigen” can be understood as those antigens presented on tumor cells. These antigens may be presented on the cell surface along with their extracellular components, often comprising both transmembrane and cytoplasmic portions of the molecule. These antigens may, in some cases, be presented only by tumor cells and never by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More general antigens are those presented by both tumor cells and normal cells, and these are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells or, due to the less compact structure of tumor tissue compared to normal tissue, are accessible to tumor cells for antibody binding. Non-exclusive examples of tumor antigens used herein include CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, BCMA, and PSMA.

[0258] Further target cell surface antigens specific to immunological impairment in this context include, for example, TL1A and TNF-alpha. The targets are preferably addressed by bispecific antigen-binding molecules, which are preferably full-length antibodies. In a very preferred embodiment, the antibody is a hetero-IgG antibody.

[0259] In a preferred embodiment of this antigen-binding molecule, the tumor antigen, preferably the tumor antigen, is selected from the group consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, CLDN6, STEAP 1, BCMA, and PSMA.

[0260] In one embodiment, the antigen-binding molecule is arranged in the order of amino to carboxyl, (a) Sequence numbers 7, 8, 17, 27, 28, 37, 38, 39, 40, 41, 48, 49, 50, 51, 52, 59, 60, 61, 62, 63, 64, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93, 100, 101, 102, 103, 104, 113, 114, 121, 122, 123, 124, 125, 131, 132, 133, 134, A first domain having 80, 90, 95, 99, or 100% sequence identity with an amino acid sequence selected from the group consisting of 135, 136, 143, 144, 145, 146, 147, 148, 149, 150, 151, 158, 159, 160, 161, 162, 163, 164, 165, 166, 173, 174, 175, 176, 177, 178, 179, 180, 181, 223, 235, and 246; (b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 187-189; c A second domain having an amino acid sequence selected from the group consisting of 80, 90, 95, 99, or 100% sequence identity with any of the sequence numbers SEQ ID NOs. 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187, or SEQ ID NOs. 26, 382, ​​and 383, as described in International Publication No. 2008 / 119567; (d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) A first polypeptide monomer of a third domain having 80, 90, 95, 99, or 100% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NOs: 437-444; (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) A second polypeptide monomer of a third domain having 80, 90, 95, 99, or 100% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NOs: 437-444. Includes.

[0261] In one embodiment, the TCE molecule is understood herein to be a bispecific antigen-binding molecule, characterized by having an amino acid sequence selected from the group consisting of the following, and being for each target cell surface antigen: (a) Sequence IDs 27, 28, 37-41; CD33 (b) Each of sequence numbers 48-52; EGFRvIII (c) Each of sequence numbers 59-64; MSLN (d) Each of sequence numbers 71-82; CDH19 (e) Each of sequence numbers 100 to 104; DLL3 (f) Sequence IDs 7, 8, 17, 113 and 114; CD19 (g) Each of sequence numbers 89-93; FLT3 (h) Each of sequence numbers 121-125; CDH3 (i) Each of sequence numbers 132-136; BCMA (j) PSMA for each of sequence numbers 143-151, 158-166 and 173-181 (k) Sequence ID 213 MUC17 (l) Sequence IDs 225 and 237, respectively; CLDN18.2, and (m) Sequence ID 248 CD70 (n) Sequence ID 255 CDH3 and MSLN (o) Sequence IDs 373-375 STEAP1 (each molecule contains three chains) 373, 376 and 380; and 377-379. (p) Sequence ID 430 CLDN6

[0262] This disclosure further provides polynucleotide / nucleic acid molecules encoding antigen-binding molecules of the present invention. Polynucleotides are biomacromolecules composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides having different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. Nucleic acid molecules or polynucleotides can be double-stranded and single-stranded, linear and cyclic. Preferably, they are contained within a vector contained in a host cell. The host cell can then express the antigen-binding molecule, for example, after being transformed or transfected with the vector or polynucleotide. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0263] The genetic code is a set of rules that translate information encoded within genetic material (nucleic acids) into proteins. Biological decoding in living cells is carried out by ribosomes, which use tRNA molecules—which carry amino acids and read three nucleotides from mRNA at once—to link the amino acids in the order specified by the mRNA. This code defines how a sequence of three nucleotides, called a codon, specifies the next amino acid to be added during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies one amino acid. Because most genes are encoded with the exact same code, this particular code is often referred to as the reference genetic code or standard genetic code. While the genetic code determines the protein sequence of a given coding region, other genomic regions can influence when and where these proteins are produced.

[0264] Furthermore, this disclosure provides vectors comprising polynucleotide / nucleic acid molecules. A vector is a nucleic acid molecule used as a medium for transferring (foreign) genetic material into cells. The term “vector” includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Generally, genetically engineered vectors include an origin of replication, a multicloning site, and a selection marker. The vector itself is generally a nucleotide sequence, generally a DNA sequence, containing an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Modern vectors may include additional features in addition to the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically for the expression of a transgene in target cells and generally contain regulatory sequences.

[0265] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of an operablely linked coding sequence in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome-binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0266] Nucleic acids are "operably linked" if they have a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide if it is expressed as a protein precursor involved in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretion leader, contiguous and within the read frame. Enhancers, however, do not need to be contiguous. Linking is done by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used, according to conventional practice.

[0267] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. This term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe the viral transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves creating transient pores or "holes" in the cell membrane to allow material uptake. Transfection can be performed using calcium phosphate, by electroporation, by compressing the cell, or by mixing cationic lipids with liposome-forming substances, fusing them with the cell membrane, and accumulating internal cargo.

[0268] The term "transformation" is used to describe the nonviral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells, including plant cells. Therefore, transformation is a genetic modification of a bacterial or non-animal eukaryotic cell resulting from direct uptake from its periphery across the cell membrane and subsequent integration of exogenous genetic material (nucleic acid molecules). Transformation can be induced by artificial means. For transformation to occur, the cell or bacterium must be in a competent state where transformation can occur as a timed response to environmental conditions such as starvation and cell density.

[0269] Furthermore, this disclosure provides host cells transformed or transfected with polynucleotide / nucleic acid molecules or vectors. As used herein, the terms “host cell” or “recipient cell” are intended to include any individual cell or cell culture that may or may have been a recipient of the vectors, exogenous nucleic acid molecules and polynucleotides encoding the antigen-binding molecules disclosed herein; and / or the antigen-binding molecules themselves. The introduction of each substance into a cell is carried out by transformation, transfection, etc. The term “host cell” is also intended to include single-cell offspring or potential offspring. In subsequent generations, certain modifications may occur due to spontaneous, accidental, or intentional mutations, or due to environmental influences, and such offspring may not actually be completely identical to the parent cell (morphologically or with respect to the genome or total DNA set), but are still included within the scope of the terms as used herein. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells.

[0270] Antigen-binding molecules can be produced within bacteria. After expression, these antigen-binding molecules can be isolated from E. coli cell paste in the soluble fraction and purified, for example, by affinity chromatography and / or size exclusion chromatography. Final purification can be carried out, for example, in the same manner as the purification process for antibodies expressed in CHO cells.

[0271] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for the antigen-binding molecules of the present invention. Saccharomyces cerevisiae or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, several other genera, species, and strains are generally available and useful in the present invention, for example, Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), and K. drosophilarum (ATCC 16045). Hosts of the genus Kluyveromyces, such as K. thermotolerans and K. marxianus (36906); yarrowia (European Patent No. 402226); Pichia pastoris (European Patent No. 183070); Candida; Trichoderma reesia (European Patent No. 244234); Neurospora crassa; Schwanniomyces occidentalis Hosts include the genus Schwanniomyces (such as Schwanniomyces occidentalis), as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger.

[0272] Suitable host cells for the expression of glycosylated antigen-binding molecules are those derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding acceptable insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. Various viral strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are officially available and can be used as the viruses herein, particularly for the transfection of Spodoptera frugiperda cells.

[0273] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis thaliana, and tobacco can also be used as hosts. Cloning and expression vectors useful for protein production in plant cell cultures are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78, Owen et al. (1992) Bio / Technology 10:790-794, Artsaenko et al. (1995) The Plant J 8:745-750, and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0274] However, there is the greatest interest in vertebrate cells, and the proliferation of vertebrate cells under culture (tissue culture) conditions has become a standard procedure. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, 1413 8065); Mouse mammary tumor cells (MMT 060562, ATCC CCL 5 1); TRI cells (Mather et al, Annals NY Acad.Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and Human hepatocellular carcinoma cell line (Hep G2).

[0275] In further embodiments, the disclosure provides a step for producing an antigen-binding molecule, comprising culturing host cells under conditions that enable the expression of the antigen-binding molecule, and recovering the generated antigen-binding molecule from the culture.

[0276] As used herein, the term “culture” refers to the maintenance, differentiation, growth, proliferation, and / or propagation of cells in vitro under favorable conditions in a culture medium. The term “expression” includes, but is not limited to, any stage involved in the production of antigen-binding molecules, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0277] When using recombinant technology, antigen-binding molecules can be produced in the perimembranous space within cells or secreted directly into the culture medium. If antigen-binding molecules are produced intracellularly, the first step is to remove granular fragments of host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the perimembranous space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell fragments can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). To inhibit protein degradation, protease inhibitors such as PMSF may be included in any of the aforementioned steps, and antibiotics may be included to prevent the growth of exogenous contaminating bacteria.

[0278] Antigen-binding molecules prepared from host cells can be recovered or purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Depending on the recovered antibody, other protein purification techniques may be used, such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, and heparin EPHAROSE. (商標)Chromatography on the above surface, chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatographic focusing SDS-PAGE, and ammonium sulfate precipitation are also available. If the antigen-binding molecule contains a CH3 domain, Bakerbond ABX resin (JTBaker, Phillipsburg, NJ) is useful for purification.

[0279] Affinity chromatography is a preferred purification technique. The matrix to which affinity ligands bind is almost always agarose, but other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than those achievable with agarose.

[0280] Furthermore, this disclosure provides a pharmaceutical composition comprising an antigen-binding molecule or an antigen-binding molecule prepared according to the process disclosed herein. The uniformity of the antigen-binding molecule in the pharmaceutical composition is preferably ≥80%, more preferably ≥81%, ≥82%, ≥83%, ≥84%, or ≥85%, even more preferably ≥86%, ≥87%, ≥88%, ≥89%, or ≥90%, still even more preferably ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%, and most preferably ≥96%, ≥97%, ≥98%, or ≥99%.

[0281] As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions contain one or more antigen-binding molecules, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises one or more suitable formulations of pharmaceutically effective carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. The components of an acceptable composition are preferably nontoxic to the recipient at the dose and concentration employed. Pharmaceutical compositions include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0282] This composition may contain a pharmaceutically acceptable carrier. Generally, as used herein, “pharmaceutically acceptable carrier” means all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, e.g., phosphate-buffered saline (PBS) solutions, water, suspensions, emulsions such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media, and coatings that are suitable for pharmaceutical administration, and especially for parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well known conventional methods.

[0283] Certain embodiments provide pharmaceutical compositions comprising an antigen-binding molecule and one or more excipients, such as those described more exemplary in this section and elsewhere in this specification. Excipients may be used in this context for a wide range of purposes, including processes for modifying the physical, chemical, or biological properties of a formulation, such as viscosity, and / or improving its efficacy, and / or stabilizing such formulations, as well as processes for preventing degradation and damage caused by stress during, for example, manufacturing, transport, storage, preparation before use, administration, and thereafter.

[0284] In certain embodiments, the pharmaceutical composition may contain formulation materials intended to modify, sustain, or protect, for example, the pH, molar osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18” Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulation materials may include, but are not limited to, the following: • Amino acids, such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine; charged amino acids, such as lysine, lysine acetate, arginine, glutamate, and / or histidine. • Antimicrobial agents such as antibacterial and antifungal agents • Antioxidants such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite; • Buffers, buffer systems, and buffering agents used to maintain a composition at or slightly below physiological pH; examples of buffers include borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids, succinates, phosphates, and histidine; for example, Tris buffer with a pH of approximately 7.0–8.5; Non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; • Aqueous carriers such as water, alcoholic / aqueous solutions, emulsions, or suspensions, such as physiological saline and buffer media; • Biodegradable polymers such as polyester; • Fillers such as mannitol or glycine; • Chelating agents such as ethylenediaminetetraacetic acid (EDTA); • Isotonic agents and absorption retarders; • Complexing agents, such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin • Bulking agent; Monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol; • (Low molecular weight) proteins, polypeptides, or proteinaceous carriers, such as human or bovine serum albumin, gelatin, or preferably immunoglobulins of human origin; • Colorants and fragrances; • Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate. • Diluent; • Emulsifier; • Hydrophilic polymers such as polyvinylpyrrolidone); • Salt-forming counterions such as sodium; • Preservatives, such as antibacterial agents, antioxidants, chelating agents, and inert gases; examples include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide. • Metal complexes such as Zn-protein complexes; • Solvents and co-solvents (such as glycine, propylene glycol, or polyethylene glycol); • Sugars and sugar alcohols, e.g., trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myo-inositose, galactose, lactitol, ribitol, myo-inositol, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene glycol; and polyhydric sugar alcohols; • Suspending agent; • Surfactants or wetting agents, e.g., Pluronic, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyroxapole; the surfactant may be a detergent having a molecular weight of >1.2 kD and / or a polyether having a molecular weight of >3 kD; non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000; • Stabilizers such as sucrose or sorbitol; • Isotonic enhancers, such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, or sorbitol; Parenteral delivery vehicles, such as sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution, or fixative oil; • Intravenous delivery vehicles, such as body fluids, nutritional supplements, and electrolyte supplements (e.g., ringer's dextrose-based).

[0285] Various components of a pharmaceutical composition (such as those listed above) may have different effects; for example, amino acids may act as buffers, stabilizers, and / or antioxidants; mannitol may act as fillers and / or isotonic enhancers; and sodium chloride may act as delivery vehicles and / or isotonic enhancers.

[0286] In addition to the polypeptides defined herein, this composition may contain further biologically active agents depending on the intended use of the composition. Such agents may include those known in the art, such as drugs acting on the gastrointestinal system, drugs acting as cell proliferation inhibitors, drugs preventing hyperuricemia, drugs inhibiting immune responses (e.g., corticosteroids), drugs modulating inflammatory responses, drugs acting on the circulatory system, and / or cytokines. This antigen-binding molecule is also intended to be used in concurrency therapy, i.e., in combination with another anticancer drug.

[0287] In certain embodiments, the composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding molecule. In certain embodiments, the main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, suitable vehicles or carriers may be water for injection, saline solution, or artificial cerebrospinal fluid, supplemented optionally with other materials common in parenteral administration compositions. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the antigen-binding molecule of the composition may be prepared for storage by mixing a selected composition having a desired degree of purity with an optional formulation (REMINGTON'S PHARMACEUTICAL SCIENCES above) in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, the antigen-binding molecule may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0288] When parenteral administration is intended, the therapeutic compositions for use herein may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antigen-binding molecule of the present invention in a pharmaceutically acceptable vehicle. A vehicle particularly suitable for parenteral injection is sterile distilled water in which the antigen-binding molecule is formulated as a sterile isotonic solution that has been appropriately preserved. In certain embodiments, the formulation may include a formulation of the desired molecule with a drug that can provide sustained-release or sustained-release of the product that can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymer compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid having an effect of increasing the duration of action in circulation may also be used. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen-binding molecule.

[0289] Antigen-binding molecules can also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation technology or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such technologies are described in Remington's Pharmaceutical Sciences, 16. th This is disclosed in edition, Oslo, A.Ed. (1980).

[0290] Pharmaceutical compositions used for in vivo administration are generally provided as sterile formulations. Sterilization can be achieved by filtration with a sterile filtration membrane. When the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Parenteral compositions can be stored in lyophilized form or as a solution. Parenteral compositions are generally filled into containers with a sterile access port, such as intravenous solution bags or vials with stoppers that can be penetrated by subcutaneous needles.

[0291] Another embodiment includes a self-buffering antigen-binding molecule preparation that can be used as a pharmaceutical composition, as described in International Publication No. 06138181A2 (PCT / US2006 / 022599). For information on protein stabilization and useful pharmaceutical materials and methods related thereto, various explanations are available in Arakawa et al., “Solvent interactions in pharmaceutical formulations,” Pharm Res. 8(3):285-91 (1991); Kendrick et al., “Physical stabilization of proteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002); and Randolph et al., “Surfactant-protein interactions,” Pharm Biotechnol. 13:159-75 (2002). Please refer in particular to the sections concerning processes for excipients and self-buffering protein formulations, especially those concerning protein pharmaceuticals and processes for veterinary and / or human medical use.

[0292] Salts may be used according to specific embodiments to adjust, for example, the ionic strength and / or isotonicity of a formulation and / or improve the solubility and / or physical stability of the protein or other components of the composition. As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and shielding charged and polar groups in the protein, thereby reducing the intensity of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize denatured proteins, particularly by binding to denatured peptide bonds (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in the protein can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0293] Ionic species exhibit remarkably different effects on proteins. Several classifications of ions and their effects on proteins have been developed and can be used in the formulation of pharmaceutical compositions. One example is the Hofmeister series, which ranks ionic and polar nonionic solutes according to their effect on the higher-order structural stability of proteins in solution. Stabilizing solutes are called "cosmotropic." Destabilizing solutes are called "chaotropic." Cosmotropes are generally used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are generally used to denature and / or solubilize proteins ("salting out"). The relative effects of ions on "salting out" and "salting out" define the position of ions in the Hofmeister series.

[0294] Free amino acids can be used as fillers, stabilizers, and antioxidants in antigen-binding molecular formulations according to various embodiments, as well as in other standard applications. Lysine, proline, serine, and alanine can be used to stabilize proteins in formulations. Glycin is useful for ensuring proper cake structure and properties in lyophilization. Arginine may be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0295] Polyols include sugars, such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols, such as glycerol and propylene glycol, and, for the purposes of this specification, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the isotonicity of formulations. Among polyols, mannitol is useful in selected embodiments and is commonly used to ensure the structural stability of cakes in lyophilized formulations. Mannitol ensures the structural stability of cakes. Generally, it is used with lyophilization protectants, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting isotonicity and as stabilizers to protect against freeze-thaw stress during transport or bulk preparation in the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can saccharify surface lysine and arginine residues. Therefore, these are not typically included among the preferred polyols for use herein. In addition, sugars that form such reactive species, such as sucrose, are also hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation, and are therefore not included among the preferred polyols in this respect. PEG is useful for stabilizing proteins and as a cryoprotective agent, and can be used in this regard.

[0296] Embodiments of antigen-binding molecule formulations further include surfactants. Protein molecules are prone to adsorption to surfaces and denaturation and resulting aggregation at gas-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to the protein concentration. These harmful interactions are generally inversely proportional to the protein concentration and are usually aggravated by physical agitation, such as that occurring during product transport and handling. Surfactants are conventionally used to prevent, minimize, or reduce surface adsorption. Useful surfactants in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188. Surfactants are also commonly used to control the stereostructural stability of proteins. In this respect, the use of surfactants is protein-specific, as any given surfactant typically stabilizes some proteins and destabilizes others.

[0297] Polysorbates are prone to oxidative degradation and, in many cases, contain sufficient amounts of peroxides when supplied to cause oxidation of the side chains of protein residues, particularly methionine. Therefore, polysorbates should be used with caution and only at the minimum impact concentration. In this regard, polysorbates exemplify the general rule that excipients should be used at the minimum impact concentration.

[0298] Embodiments of the antigen-binding molecule of the formulation of the present invention further comprise one or more antioxidants. By maintaining appropriate levels of ambient oxygen and ambient temperature, and by avoiding exposure to light, harmful oxidation of proteins in the pharmaceutical formulation can be prevented to some extent. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Antioxidants particularly useful in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the product's shelf life. In this respect, EDTA is a preferred antioxidant according to the present invention. Antioxidants can damage proteins. For example, reducing agents, such as glutathione, can particularly break intramolecular disulfide bonds. Therefore, antioxidants for use in the present invention are selected, among other things, to eliminate or significantly reduce the possibility that they themselves may damage proteins in the formulation.

[0299] The formulation according to the present invention may contain metal ions that are cofactors of proteins and are required to form protein coordination compounds, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit several processes that degrade proteins. However, metal ions can also catalyze physical and chemical processes that degrade proteins. Magnesium ions (10-120 mM) may be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 Ions (up to 100 mM) can increase the stability of starfish oxyribonuclease. However, Mg +2 Mn +2 and Zn +2 This can destabilize rhDNase. Similarly, Ca +2 and Sr +2 It can stabilize factor VIII, which is Mg +2 Mn +2 and Zn +2 Cu +2 and Fe +2 It can be destabilized by Al, and its aggregation is+3 It can be amplified by ions.

[0300] Embodiments of the antigen-binding molecule of the formulation of the present invention further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations that require two or more dispensings from the same container. Their main function is to inhibit microbial growth over the shelf life or shelf life of the formulation and ensure the sterility of the product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use with low-molecular-weight parenteral drugs, the development of protein formulations containing preservatives can be challenging. Preservatives almost always have an destabilizing effect (aggregation) on proteins, which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs are formulated only for single use. However, the availability of multi-dose formulations offers advantages in terms of patient convenience and high marketability. Human growth hormone (hGH), for which the development of preservative-treated formulations has led to the commercialization of more convenient multi-dose injectable pens, is a good example. At least four such pen devices containing hGH-preserved formulations are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized - dual-chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol.

[0301] As expected, developing liquid formulations containing preservatives is more challenging than developing lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with preservative-containing diluents at the time of use. This reduces the time the preservative is in contact with proteins, significantly minimizing the associated stability risks. In the case of liquid formulations, the effectiveness and stability of the preservative should be maintained throughout the entire product shelf life (approximately 18-24 months). An important point to note is that the effectiveness of the preservative must be demonstrated in the final formulation containing the active drug and all excipient components.

[0302] The antigen-binding molecules disclosed herein may also be formulated as liposomes. “Liposomes” are small vesicles composed of various types of lipids, phospholipids, and / or surfactants useful for drug delivery to mammals. The components of liposomes are generally arranged in a bilayer structure, similar to the arrangement of lipids in biological membranes. Liposomes containing antigen-binding molecules are prepared by methods known in the art, such as those described in, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and International Publication No. 97 / 38731. Liposomes with extended circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be prepared by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). To obtain liposomes with a desired diameter, the liposomes are extruded through a filter of a predetermined pore size. The Fab' fragment of the antigen-binding molecule of the present invention can be compounded into liposomes via a disulfide exchange reaction, as described in Martin et al. J. Biol. Chem. 257:286-288 (1982). Optionally, chemotherapeutic agents are contained within the liposomes. See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

[0303] After the pharmaceutical composition has been formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as an aerosolized or lyophilized powder. Such formulations may be stored in a form ready for immediate use or in a form that is reconstituted before administration (e.g., lyophilized product).

[0304] The biological activity of the pharmaceutical compositions as defined herein can be determined, for example, by cytotoxic assays as described in the following examples in International Publication No. 99 / 54440 or in Schlereth et al. (Cancer Immunol.Immunother.20(2005),1-12). As used herein, “efficacy” or “in vivo efficacy” refers to the response to therapy with the pharmaceutical compositions of the present invention, for example, using standardized NCI response criteria. The success or in vivo efficacy of therapy using the pharmaceutical compositions of the present invention refers to the efficacy of the composition with respect to its intended purpose, i.e., the composition’s ability to cause its desired effect, i.e., a drastic reduction in diseased cells, such as tumor cells. In vivo efficacy can be observed by established standard methods for each disease entity, including, but not limited to, leukocyte counting, differential counting, fluorescence-activated cell sorting, and bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used.Furthermore, computed tomography, X-ray, and magnetic resonance imaging (e.g., response evaluation based on National Cancer Institute criteria) [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999] Positron emission tomography (Apr;17(4):1244)), leukocyte counting, differential counting, fluorescence-activated cell sorting, bone marrow aspiration, lymph node biopsy / histology, and clinical chemistry parameters specific to various lymphomas (e.g., lactate dehydrogenase), as well as other established standard methods may be used.

[0305] Another major challenge in the development of drugs, such as the pharmaceutical compositions of the present invention, is the predictable regulation of pharmacokinetic properties. For this purpose, a pharmacokinetic profile of a candidate drug, i.e., a profile of pharmacokinetic parameters that influence a particular drug's ability to treat a given disease, can be established. Pharmacokinetic parameters of a drug that influence a drug's ability to treat certain diseases include, but are not limited to, half-life, volume of distribution, first-pass metabolism in the liver, and degree of serum binding. The efficacy of a given drug may be influenced by each of the above parameters. A hypothetical feature of the antigen-binding molecules of the present invention, brought about by a specific FC (Functional Cell) configuration, is that they include, for example, differences in pharmacokinetic behavior. It is preferable that the targeted antigen-binding molecules with extended half-lives according to the present invention exhibit a remarkably increased residence time in vivo compared to the "canonical" non-HLE version of the antigen-binding molecule.

[0306] "Half-life" refers to the time it takes for 50% of an administered drug to be eliminated through biological processes such as metabolism and excretion. "First-pass metabolism in the liver" refers to the tendency of a drug to be metabolized upon initial contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" refers to the degree to which a drug is retained in various compartments of the body, such as intracellular and extracellular spaces, tissues, and organs, as well as the distribution of the drug within these compartments. "Degree of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins such as albumin, leading to a reduction or loss of the drug's biological activity.

[0307] Pharmacokinetic parameters include bioavailability, lag time (Tlag), Tmax, absorption rate, and / or Cmax for a given amount of drug administered. "Bioavailability" refers to the amount of drug in the blood compartment. "Lag time" refers to the time delay between drug administration and its detection and measurability in the blood or plasma. "Tmax" is the time to reach the peak blood concentration of the drug, and "Cmax" is the peak blood concentration achievable by a given drug. The time it takes for a drug to reach the blood or tissue concentration required for its biological effect is influenced by all parameters. As outlined above, the pharmacokinetic parameters of bispecific antigen-binding molecules exhibiting interspecies specificity, which can be determined in preclinical animal studies in primates other than chimpanzees, are also shown, for example, in the publication by Schlereth et al. (Cancer Immunol.Immunother.20(2005),1-12).

[0308] In a preferred embodiment of the present invention, the pharmaceutical composition is stable at approximately -20°C for at least 4 weeks. As is evident from the accompanying examples, the quality of the antigen-binding molecule of the present invention, compared to the quality of the corresponding state-of-the-art antigen-binding molecule, can be tested using different systems. These tests are understood to conform to the "ICH Harmonised Tripartite Guideline: Stability Testing of Biotechnological / Biological Products Q5C and Specifications: Test procedures and Acceptance Criteria for Biotechnological / Biological Products Q6B," and are selected to provide a stability index profile that ensures reliable detection of changes in the identity, purity, and capacity of the product. It is well accepted that the term purity is a relative term. Due to the effects of glycosylation, deamidation, or other heterogeneity, the absolute purity of a biotechnological / biological product should usually be assessed by two or more methods, and the resulting purity values ​​are method-dependent. For the purpose of stability testing, purity testing should be aligned with the method for determining degradation products.

[0309] To evaluate the quality of the pharmaceutical composition containing the antigen-binding molecule of the present invention, it may be analyzed, for example, by analyzing the content of soluble aggregates (HMWS by size exclusion) in the solution. The stability at about -20°C for at least 4 weeks is preferably characterized by an HMWS content of less than about 5%, more preferably less than 2.5%, and even more preferably less than 1.5%.

[0310] Further examples of evaluating the stability of antigen-binding molecules in the form of pharmaceutical compositions are provided in the attached Examples 4–12. In these examples, embodiments of antigen-binding molecules are tested against different stress conditions in different pharmaceutical formulations, and the results are compared with other bispecific T-cell engagement antigen-binding molecules in extended half-life (HLE) forms known from the Art. Generally, antigen-binding molecules provided in a particular FC form according to the present invention are assumed to be more stable against a wide range of stress conditions, such as temperature and light stress, compared to antigen-binding molecules provided in different HLE forms and antigen-binding molecules that do not have any HLE form (e.g., "canonical" antigen-binding molecules). The temperature stability may relate to both low temperatures (below room temperature, including freezing temperatures) and high temperatures (above room temperature, including temperatures up to or above body temperature). As those skilled in the art will recognize, such improved stability against stresses that are difficult to avoid in clinical practice makes the antigen-binding molecule safer because it results in fewer degradation products in clinical practice. Consequently, the improved stability means improved safety.

[0311] One embodiment provides an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the method of the present invention for use in the prevention, treatment, or remission of proliferative disorders, neoplastic diseases, viral diseases, or immunodeficiencies.

[0312] The formulations described herein are useful as pharmaceutical compositions for treating, improving and / or preventing the pathological medical conditions described herein in patients in need thereof. The term “treatment” refers to both therapeutic treatments and preventive or protective measures. Treatment includes the application or administration of formulations to the body, isolated tissues or cells of a patient having a disease / disorder, symptoms of a disease / disorder, or predisposition to a disease / disorder, with the aim of curing, resolving, reducing, alleviating, altering, correcting, relieving, improving, or influencing the disease, symptoms of a disease or predisposition to a disease.

[0313] As used herein, the term “remission” means improvement in the disease state of a patient with a tumor, cancer, or metastatic cancer as described below herein, by administering the antigen-binding molecule according to the present invention to a target requiring its use. Such improvement may also be considered as a slowing or cessation of the progression of the patient’s tumor, cancer, or metastatic cancer. As used herein, the term “prevention” means avoidance of the onset or recurrence of a patient with a tumor, cancer, or metastatic cancer as described below herein, by administering the antigen-binding molecule according to the present invention to a target requiring its use.

[0314] The term “disease” refers to any condition that would benefit from treatment with the antigen-binding molecules or pharmaceutical compositions described herein. This term includes chronic and acute disorders or diseases, including pathological conditions that make mammals susceptible to the disease in question.

[0315] A "neoplasm" is an abnormal growth of tissue, which, while not always, usually forms a mass. When it forms a mass, it is generally called a "tumor." Neoplasms or tumors can be benign, occult malignant (precancerous), or malignant. Malignant neoplasms are generally called cancer. They can usually invade and destroy surrounding tissues, forming metastases, that is, they spread to other parts, tissues, or organs of the body. Therefore, the term "metastatic cancer" includes not only metastases from the primary tumor but also metastases to other tissues or organs. Lymphomas and leukemias are lymphoid neoplasms. For the purposes of this invention, they are also included in the terms "tumor" or "cancer."

[0316] The term "viral disease" refers to a disease resulting from an infection with a particular virus.

[0317] As used herein, the term “immune disorder” refers to immune disorders such as autoimmune diseases, hypersensitivity, and immunodeficiency, in accordance with the general definition of the term.

[0318] In one embodiment, the present invention provides a method for treating or relieving a proliferative disorder, a neoplastic disorder, a viral disorder, or an immunodeficiency, comprising the step of administering an antigen-binding molecule of the present invention or an antigen-binding molecule prepared according to the method of the present invention to a subject in need thereof.

[0319] The terms “subjects in need” or “subjects in need of treatment” include subjects who already have the disorder and subjects for whom the disorder will be prevented. Subjects in need or “patients” include human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0320] The molecules of the present invention are generally designed for specific routes and methods of administration, specific dose-to-frequency administration, and specific treatments of specific diseases, particularly in terms of bioavailability and persistence. The materials of the composition are preferably formulated at concentrations acceptable at the administration site.

[0321] Accordingly, formulations and compositions can be designed in accordance with the present invention to be delivered by any preferred route of administration. In relation to the present invention, possible routes of administration include: • Local routes (e.g., on the skin, by inhalation, nose, eyes, auricle / ear, vagina, mucous membranes); • Intestinal pathways (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and • Parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extraamniotic, intraarticular, intracardiac, intradermal, intrafocal, intrauterine, intrabladder, intravitreous, percutaneous, intranasal, transmucosal, synovial bursa, intraluminal) These are some examples, but are not limited to them.

[0322] The pharmaceutical composition and antigen-binding molecule of the present invention are particularly useful for parenteral administration, such as subcutaneous or intravenous delivery, by injection, such as bolus injection, or by infusion, such as continuous infusion. The pharmaceutical composition may also be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0323] In particular, the present invention enables the uninterrupted administration of a preferred composition. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e., continuous administration can be achieved by a patient-worn miniature pump system for regulating the inflow of the therapeutic agent into the patient's body. Pharmaceutical compositions comprising the antigen-binding molecules of the present invention can be administered by using such a pump system. Such pump systems are generally known in the art and typically involve the periodic replacement of a cartridge containing the therapeutic agent to be injected. When a cartridge is replaced in such a pump system, a temporary interruption may occur in the inflow of the therapeutic agent into the patient's body, which is otherwise uninterrupted. Even in such cases, the administration stage before and after cartridge replacement will still be considered within the meaning of the pharmaceutical means and methods of the present invention, which together constitute the “uninterrupted administration” of such therapeutic agent.

[0324] The continuous or uninterrupted administration of the antigen-binding molecule of the present invention may be intravenous or subcutaneous administration by a fluid delivery device or a small pump system comprising a fluid delivery mechanism for delivering fluid from a reservoir and a drive mechanism for driving the delivery mechanism. A pump system for subcutaneous administration may include a needle or cannula that penetrates the patient's skin and delivers the preferred composition into the patient's body. The pump system can be directly fixed or attached to the patient's skin, whether vein, artery, or blood vessel, to allow direct contact between the pump system and the patient's skin. The pump system can be attached to the patient's skin for 24 hours to several days. There may also be small pump systems with a small reservoir volume. In non-limiting examples, the volume of the reservoir of the preferred pharmaceutical composition to be administered may be 0.1 to 50 ml.

[0325] Continuous administration may also be performed transdermally by patches that are applied to the skin and replaced at intervals. Those skilled in the art are aware of patch systems for drug delivery suitable for this purpose. It should be noted that transdermal administration is particularly suitable for uninterrupted administration because, for example, a new second patch can be applied to the skin surface directly adjacent to the first used patch, immediately before the first used patch is removed, and the replacement of the first used patch can be completed at the same time. There are no problems with inflow interruption or battery failure.

[0326] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with an appropriate liquid before administration. The lyophilized material may be reconstituted with, for example, bacteriostatic water for injection (BWFI), physiological saline, phosphate-buffered saline (PBS), or the same formulation in which the protein was before lyophilization.

[0327] The composition of the present invention can be administered to a subject in a suitable dose, which can be determined, for example, by a dose-escalation study in which the antigen-binding molecule of the present invention exhibiting interspecies specificity as described herein is administered to a primate other than a chimpanzee, such as a macaque, in increasing doses. As described above, the antigen-binding molecule of the present invention exhibiting interspecies specificity as described herein has the advantage of being usable in the same form in preclinical studies of primates other than chimpanzees and being usable as a drug in humans.

[0328] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The term “therapeutic effective dose” is defined as the amount sufficient to cure, or at least partially suppress, the disease and its complications in a patient who already has the disease. The amount or dose that is effective for this use depends on the condition being treated (indication), the antigen-binding molecule being delivered, the nature and purpose of the treatment, the severity of the disease, prior treatment, the patient’s medical history and response to the treatment, the route of administration, body size (weight, body surface area, or organ size), and / or the patient’s condition (age and overall health), as well as the patient’s overall immune system status.

[0329] A therapeutically effective amount of the antigen-binding molecule of the present invention preferably reduces the severity of disease symptoms, increases the frequency or duration of disease-free periods, or prevents functional impairment or disability resulting from the suffering of the disease. With regard to the treatment of antigen-expressing tumors on target cells, a therapeutically effective amount of the antigen-binding molecule of the present invention, such as an anti-target cell antigen / anti-CD3 antigen-binding molecule, preferably inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of compounds to inhibit tumor growth can be evaluated in animal models to predict efficacy.

[0330] This pharmaceutical composition may be administered in a single treatment, or, as necessary, in combination with additional treatments such as anticancer therapy, for example, other protein-based and non-protein-based drugs. These drugs may be administered simultaneously with the composition containing the antigen-binding molecule of the present invention as defined herein, or they may be administered separately at predetermined time intervals and doses before or after the administration of the antigen-binding molecule.

[0331] As used herein, the term “effective and non-toxic dose” means a tolerable dose of the antigen-binding molecule of the present invention that is sufficiently high to result in a drastic reduction of diseased cells, tumor disappearance, tumor regression, or stabilization of the disease without causing or essentially causing serious toxic effects. Such an effective and non-toxic dose may be determined, for example, by dose-escalation studies described in the Art, and this dose must be below the dose that induces a serious adverse side effect (dose-limiting toxicity, DLT).

[0332] As used herein, the term “toxicity” refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These adverse events may refer to systemic drug intolerance and / or local intolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0333] As used herein, the terms “safety,” “in vivo safety,” or “tolerability” are defined as the administration of a drug that does not induce serious adverse events immediately after administration (local tolerability) and during longer periods of drug use. “Safety,” “in vivo safety,” or “tolerability” can be assessed periodically, for example, during treatment and follow-up. Measurements include clinical assessments, e.g., screening for organ findings and abnormal clinical laboratory values. Clinical assessments may be performed, and deviations from normal findings may be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings may include, for example, criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmias, and coagulation, as shown in the Common Terminology Criteria for adverse events v3.0 (CTCAE). Laboratory parameters that may be tested include, for example, hematology, clinical chemistry, coagulation profiles, and urinalysis, as well as tests of other body fluids, e.g., serum, plasma, lymph, or cerebrospinal fluid. Therefore, safety can be evaluated, for example, by measuring examination parameters and recording adverse events through physical examination, imaging techniques (i.e., ultrasound, X-ray, CT scan, magnetic resonance imaging (MRI), other measurements using technical devices (i.e., electrocardiogram), and vital signs. For example, in the use and methods according to the present invention, adverse events in primates other than chimpanzees may be tested by histopathological and / or histochemical methods.

[0334] The above terms are also referenced, for example, in the Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting on July 16, 1997.

[0335] Finally, the present invention provides a kit comprising the antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the steps of the present invention, the pharmaceutical composition of the present invention, the polynucleotide of the present invention, the vector of the present invention, and / or the host cell of the present invention.

[0336] In relation to the present invention, the term "kit" means two or more components packaged together in a container, vessel, or other means, where one of the components corresponds to an antigen-binding molecule, pharmaceutical composition, vector, or host cell of the present invention. Accordingly, a kit can be described as a set of products and / or tools sufficient to achieve a particular purpose, which can be sold individually.

[0337] The kit may include one or more containers of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing an appropriate dosage (see above) of the antigen-binding molecule or pharmaceutical composition of the present invention for administration (see above). The kit may further include instructions for use (e.g., in the form of a leaflet or instruction manual), means for administering the antigen-binding molecule of the present invention such as a syringe, pump, or infuser, means for reconstituting the antigen-binding molecule of the present invention, and / or means for diluting the antigen-binding molecule of the present invention.

[0338] The present invention also provides kits for single-dose administration units. The kits of the present invention may also comprise a first container containing a dried / lyophilized antigen-binding molecule and a second container containing an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0339] Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise explicitly indicated by the context. For example, a reference to “reagent” includes one or more such reagents, and a reference to “method” includes references to equivalent steps and methods known to those skilled in the art, which may be modified for or substituted for the methods described herein.

[0340] Unless otherwise specified, the term “at least” preceding a set of elements should be understood to refer to all elements within that set. Those skilled in the art will recognize, or can verify through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be incorporated herein.

[0341] Wherever the terms “and / or” are used in this specification, they include the meanings of “and,” “or,” and “any other combination of the elements connected by the terms.”

[0342] As used herein, the terms “about” or “approximately” mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, the term also includes specific numbers; for example, “about 20” includes 20.

[0343] The terms "less than" or "greater than" include specific numbers. For example, "less than 20" means less than or equal to 20. Similarly, "greater than" or "greater than" means greater than or equal to, or greater than or equal to, respectively.

[0344] Throughout this specification and the subsequent claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” mean to include the integer or process or group of integers or processes described, but not to exclude any other integer or process or group of integers or processes. As used herein, the term “omprising” may also be replaced by the terms “containing” or “including,” or, as used herein, sometimes by the term “having.”

[0345] As used herein, “consisting of” excludes any component, process or ingredient not specified in the claim. As used herein, “essentially consisting of” does not exclude materials or processes that do not substantially affect the basic and novel features of the claim.

[0346] In each example herein, the terms “contains,” “essentially consist of,” and “consist of” may be replaced with one of the other two terms.

[0347] It should be understood that the present invention is not limited to, but may vary from, the specific methodologies, protocols, materials, reagents, and substances described herein. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is defined solely by the claims.

[0348] All publications and patents (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) referenced throughout this specification, whether above or below, are incorporated herein by reference in their entirety. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosures by prior invention. This specification takes precedence over any such material incorporated by reference unless such material is inconsistent with or in agreement with this specification.

[0349] A better understanding of the present invention and its advantages can be obtained from the following examples, but these examples are provided for illustrative purposes only. These examples are not intended in any way to limit the scope of the present invention. [Examples]

[0350] Example 1: Binding peptides for binding peptide modification and polymer complex formation, and their characteristics evaluation. Materials and methods for all experiments: Peptide-carrier compounding: Disulfide complex formation reaction: reagent: Reaction buffer: PBS 100mM phosphate, 250mM NaCl, pH 6.5, filtered (endotoxin-free) cell culture water 15mL Falcon tube Amicon Filtration Centrifugal Tubing

[0351] procedure: Dissolve OPSS or maleimide-containing PEG in reaction buffer to a concentration of 4 mM. Dissolve the bound peptide in cell culture water or reaction buffer to a concentration of 5 mM. The reaction was carried out using 62.5 μM to 125 μM PEG in a total volume of 7200 μL. Add PEG and reaction buffer to a 10 mL Falcon tube. Slowly add 8 equivalents of peptide while gently vortexing. Rotate the mixture at room temperature for 3 hours. After 3 hours, the reaction mixture is added to an appropriately sized MW amicon filtration unit (i.e., a 3kDa filter for complexes derived from 4-arm 2- and 5-kDa PEG, and a 10-kDa filter for complexes derived from 4-arm 10- and 20-kDa PEG). The reaction buffer is added until the volume reaches 15 mL, and the reaction mixture is concentrated to 1-2 mL. The concentration is repeated for the addition of buffer and a total of four concentration steps. The complexes are stored at -20°C until the concentration can be measured via a mass spectrometry quantitative assay. The reagents are stored in the reaction buffer at a concentration of 3-25 mg / mL.

[0352] Thioether complexing reaction (Mal, AcBr): 1. Reduce OPSS- or thiol-functionalized PEG with TCEP. 2. Remove TCEP (if necessary) and replace the buffer with the reaction buffer (50 mM sodium phosphate, 2 mM EDTA, pH 7.5 or equivalent). 3. Add an excess amount of BrAc-functionalized peptide. Incubate over RT. 4. Purify by RP-HPLC.

[0353] Azide / alkyne cycloaddition: 73136 pE-DGNEELKGK(mPEG)-OH, linear, 20 kDa, representative preparation of acetamide The conjugated peptide solution was first prepared by dissolving pE-DGNEELKGK(bromoacetyl)-OH at 5 mg / mL in reaction buffer 1 (50 mM phosphate, 2 mM EDTA, pH 7.5). 20 kDa α-mercaptoethyl-ω-methoxy and polyoxyethylene (SUNBRIGHT ME-200SH, NOF America, White Plains, NY) were added to a 15 mL conical tube. The peptide solution was added to a tube containing PEG to achieve a final ratio of 2 equivalents of bromoacetyl-peptide to thiol. The mixture was gently pulsed and vortexed until the PEG was completely dissolved. The pH of the solution was adjusted to 8 by adding a sufficient amount of 1 M HEPES, pH 8.5. The reaction mixture was incubated overnight at ambient temperature on a rotary incubator. The completed reaction product was stored at -20°C until purification.

[0354] Preparation of 79677 pE-DGEEELKGC(PEG)-NH2, 4-arm, 5kDa, MAL The peptide solution was first prepared by dissolving pE-DGEEELKGC-NH2 at a concentration of 5 mg / mL in reaction buffer 2 (100 mM sodium phosphate, 250 mM NaCl, pH 6.5) in a 50-mL conical tube. The PEG solution was then prepared by dissolving 5 kDa 4-arm PEG-MAL (PSB-453, Creative PEGWorks, Chapel Hill, NC) at a concentration of 50 mg / mL in reaction buffer 2. The PEG storage solution was maintained on ice throughout the procedure. The PEG solution was added dropwise to the peptide solution at ambient temperature with gentle, intermittent vortexing until the final ratio of 2 equivalents of thiol-peptide to maleimide was reached. The completed reaction product was stored at -20°C until purification.

[0355] General procedure for purifying TCE masking molecules The conjugated peptide-PEG complex was purified by reverse-phase high-performance liquid chromatography (IFC) using an Agilent HPLC system 1290 Infinity II with a Phenomenex Gemini 5um NX-C18 110A and a 250x30mm LC column. The compound was eluted over 45 minutes with a gradient from 0%B to 60%B at a flow rate of 30 mL / min using mobile phase A: 20 mM ammonium bicarbonate in water with 5% acetonitrile and mobile phase B: 20 ​​mM ammonium bicarbonate in water with 75% acetonitrile. The fraction containing the desired product was pooled, lyophilized, dissolved in deionized water, and filtered and sterilized using a 0.22 μm PDVF filter (Steriflip® Vacuum Tube Top filter, Millipore, Burlington MA). The solution was lyophilized again to remove all remaining ammonium bicarbonate. The product was redissolved in sterile deionized water, and the peptide content was determined by chemiluminescent nitrogen detection using Antek MultiTek (PAC, Houston, TX) according to the manufacturer's instructions. The final solution with a known peptide content was dispensed into sterile vials and lyophilized to formulate the final product as a pre-quantified dried powder.

[0356] - Binding assay SPR solution assay for competitive binding with TCE Measurement of equilibrium binding affinity using MinExA 1. Preparation of NHS-activated Sepharose fast-flow beads: 2. The Fast Flow Sepharose suspension in 100% IPA was washed twice with milliQ H2O, gently centrifuged, and the beads were pooled between washes. 3. Equilibrium was achieved in 0.15M NaBO3 buffer solution at pH=8.5. 4. 80 mM PDEA was added, and the resin for thiol exchange was activated at rt for 10 minutes. 5. Resin equilibrated with Hepes buffer-P (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% v / v Surfactant P2) and pH 7.4 binding buffer. 6. Add 30 ug / mL of the conjugated peptide, QDGNEEMGC (SEQ ID NO: 297) or pGlu-DGNEEMGKGMEENGDQC (SEQ ID NO: 300), and allow to react for 1 hour to functionalize the resin. 7. Quenched with 10mM L-Cys for 1 hour. 8. The constant binding partner was FLT3xCD3 TCE (SEQ ID NO: 93), MW 105720. 3.2 mg / mL 31.21 μM was diluted to 333 pM, and pGlu-DGNEEMGKGMEENGDQC peptide or pGlu-DGNEEMGKGMEENGDQC-PEG (Pep-PEG v2) was titrated to concentrations of 1 μM or 400 nM to 2.3 pM relative to the effective peptide molar concentration.

[0357] Meeting kinetics using KinExA result In vitro binding affinity and kinetics

[0358] [Table 15]

[0359] As can be seen from the results above, in this case, functionalization of the binding peptide to the original CD3 epitope in the N-terminal CD3ε QDGNEEMG (SEQ ID NO: 262) results in a lower Kd value, i.e., higher affinity, which is generally desirable.

[0360] Furthermore, Table 6 below shows the results of a surface plasmon resonance competitive binding assay that highlights the improved affinity in comparison to the physiological CD3ε epitope containing the sequence QDGNEEMG, which is achieved through the substitution of Q(Gln) to pE(pGlu) and maintained by the conserved residue substitution. As it can be derived from Table 6,

[0361] [Table 16]

[0362] Example 2: Mouse pharmacokinetic study to evaluate in vivo PK To evaluate the extension of peptide half-life through PEG-peptide complexation, the dimer and palindromic cysteine-containing peptide QDGNEEMGKGMEENGDQC, which contains lysine (assumed to be the trypsin-cleavable handle) and is manipulated in a trypsin-cleavable handle for analytical mass spectrometry quantification, was selected for the initial evaluation in mice. The 4-arm 20kDa PepPOL complex was administered IV to BL / 6 mice at dose levels of 0.82 or 8.2 mg / kg. Two uncomplexed parent peptide forms, namely QDGNEEMGKGMEENGDQ and QDGNEEMGKGMEENGDQC, were also administered at 1 mg / kg as control arms to evaluate half-life extension. The PK profile for each peptide was evaluated by quantifying the plasma concentration of the intact total peptide substance over time (see Methods). Based on curve fitting to the final slope in log-linear plot form, the final efflux half-life of the PepPOL complex was approximated to be 88 minutes. On the other hand, uncomplexed peptides showed a final elimination half-life of 3–10 minutes, which is consistent with their smaller and higher estimated renal clearance levels (see Figure 4).

[0363] Example 3: Second の Mouse PK test To evaluate the plasma pharmacokinetics of the masking peptide in the 8-arm (octavalent), 40kDa PEG form, a second PK study was performed in BL / 6 mice. Both CD3ε PepPOL, v1 (40kDa) and CD3ε PepPOL, v1 (20kDa) (see Table 6 for sequences) were administered to mice at a dose level of 5 mg / kg via IV bolus, and the results are shown in Figure XX. Interestingly, the plasma PK of CD3ε PepPOL, v1 (20kDa) administered as a control arm in this study was similar to that of the larger CD3ε PepPOL, v1 (40kDa) form, suggesting that the clearance mechanism determining the rate may be altered by the loss of the peptide and non-intact CD3ε PepPOL. The final elimination half-life was estimated to be 90 to 120 minutes for the 20kDa and 40kDa CD3ε PepPOL, v1 forms, respectively.

[0364] Example 4: TCE masking molecules in PK mouse tests To further understand the in vivo behavior of the pyro-Glu-containing dimer p-EDGNEEMGKGMEENGDQC PepPOL complex, a 4-arm 20kDa disulfide-coupled form ((pE-DGNEEMGKGMEENGDQC)4-PEG(20kDa)) (CD3ε PepPOL, v2) was synthesized and evaluated in a single-dose mouse PK test (C57BL / 6). To illustrate their similar behavior, the PK profile of the CD3ε PepPOL, v2 form is compared with that of the original CD3ε PepPOL, v1. The efflux half-life was determined by nonlinear one-phase decay fitting and estimated to be 102 minutes.

[0365] Example 5: PK evaluation of the conjugated peptide maleimide test To evaluate the effects of linker chemistry on plasma PK in rodents, monomeric p-GluDGNEELGKC peptides were coupled to 2-, 5-, 10-, and 20-kDa sized 4-arm PEG carriers via maleimide complexes, and their single-dose plasma PK was evaluated in mice. The data are plotted in Figure 6, and the PK parameters are presented in Table 3. From these studies, a size dependence of apparent distribution kinetics was observed, which is most clearly exemplified by the comparison of 2-compartment PK models that capture the degree and duration of differences in the "alpha" or distribution phase of the PK profile.

[0366] Example 6: Study of co-administration of muCD19 TCE + CD3ε PepPOL, v1 in a huCD3ε TCR KI mouse model. Mouse anti-muCD19 TCE at a dose of 100 ug / kg IV was examined at >2300X. To establish the desired PD effect using CD3ε PepPol v1, an initial pilot study was conducted in huCD3ε TCR KI mice. Here, CD3ε PepPol v1 was co-administered with 100ug / kg muCD19 TCE at a molar excess of ≥1000X (peptide:TCE). PK revealed that the TCE dose was insufficient, so the molar excess was increased to ≥1000X, resulting in a higher ratio than desired. Nevertheless, co-administration of 20kDa PepPol maintained a higher degree of PD effect in terms of CD19+ B cell depletion, while across the panel, both 20kDa and 40kDa PepPols may have an effect on cytokine reduction.

[0367] Example 7: Study of simultaneous administration of muCD19 TCE + CD3ε PepPOL,v1 in a huCD3ε TCR KI mouse model. Mouse anti-muCD19 TCE at a dose of 500 ug / kg IV + 25X, 100X and 500X molar excess CD3ε PepPOL,v1. This study aimed to investigate the characteristics of the CD3ε PepPOL,v1 dose-response in huCD3ε TCR KI mice at a planned effective muCD19 TCE dose (as determined by prior testing in a solid tumor MC38-muCD19++ model).

[0368] In short, we investigated the characteristics of the CD3ε PepPOL,v1 dose-response in huCD3ε TCR KI mice by co-administration of 500 ug / kg muCD19 TCE delivered via IV bolus injection. Pharmacodynamic endpoints included: a) CD19+ B cell immunophenotyping of both splenic and peripheral B cell populations by flow cytometry; and b) serum cytokines at 0–96 hours.

[0369] Results: No significant difference in clear B cell depletion across all dose groups was observed with CD3ε PepPOL,v1 up to a 100x molar excess in both peripheral blood and spleen. In the group administered the maximum dose of CD3ε PepPOL,v1 (500x molar excess), a small residual B cell population (approximately 2.4–6.5%) was observed in the blood and spleen 72 hours after administration. CD3ε PepPOL,v1 induced a dose-dependent decrease in all serum cytokines evaluated.

[0370] Example 8: Simultaneous administration of muCD19 TCE + CD3ε PepPOL, v1 in a huCD3ε TCR KI,MC38-muCD19++ syngeneic mouse tumor model. Mouse anti-muCD19 TCE at a dose of 500 ug / kg IV + 200X and 500X molar excesses of CD3ε PepPOL, v1. Co-administration of muCD19 TCE + CD3ε PepPOL,v1 in huCD3ε TCR KI mice harboring MC38-muCD19++ solid tumors. This study was intended to expand on the results of PD study #1 to include inhibition of solid tumor growth after a single co-administration of muCD19 + CD3ε PepPOL,v1.

[0371] Results: Complete B-cell depletion in both the spleen and blood up to 72 hours, a statistically insignificant effect on tumor growth inhibition up to day 20 in the CD3ε PepPOL,v1 combination group, and a dose-dependent decrease in all measured cytokines with CD3ε PepPOL,v1.

[0372] Example 9: Cynomolgus monkey study in which CD20 TCE (10 ug / kg) + CD3ε PepPOL, v1 (+250X (0.160 mg / kg), +750X (0.480 mg / kg) were administered simultaneously. This PK / PD trial aimed to characterize the ability of CD3ε PepPOL,v1 to desorb cytokine release from the CD20 TCE PD response (CD20+ B cell lysis) across a 3-fold dose range of CD3ε PepPOL,v1 in combination with a 10ug / kg CD20 TCE dose. The PD response was preserved in all groups, and near-complete depletion of the circulating CD20+ B lymphocyte population occurred within 24 hours post-administration. Following treatment with CD20 TCE alone, there was a mild induction of IL-2, IL-6, and MCP-1 plasma levels, peaking at 2 hours (IL-2 and IL-6) or 4 hours (MCP-1), before returning to baseline by 72 hours. The dose-response of CD3ε PepPOL,v1 was observed for MCP-1, IL-2, and IL-6 cytokines. At a maximum CD3ε PepPOL,v1 dose of 0.480 mg / kg (750X molar concentration in peptide), near-complete loss of detectable cytokines occurred at all time points.

[0373] Example 10: Cynomolgus monkey study of simultaneous administration of CD20 TCE (0.010 mg / kg) + CD3ε PepPOL, v2 (+25X (0.016 mg / kg), +250X (0.16 mg / kg)). The PK / PD trial 157130 was an exploratory dose-range study conducted in cynomolgus monkeys to establish the activity of "high affinity" CD3ε PepPOL,v2 when administered co-administered with CD20 TCE. Initial trial endpoints included: cytokine release, CD20+ B cell lysis, T cell activation, and clinical chemistry endpoints commonly associated with CRS (e.g., CRP). CD20 TCE was administered at 10 ug / kg alone, or in combination with CD3ε PepPOL,v2 at 0.016 mg / kg (25x molar excess CD3ε peptide) or 0.160 mg / kg (250x molar excess CD3ε peptide). A summary of the results is provided below.

[0374] Cytokine Serum cytokines MCP-1, IL-6, TNFα, IFNγ, and IL-2 were evaluated at multiple time points from 0 to 168 hours after administration using a multi-sandwich immunoassay.

[0375] MCP-1 Intravenous administration (IV) of 0.010 mg / kg CD20 TCE to male (n=1 / group) and female (n=1 / group) monkeys resulted in increased serum MCP-1 concentrations. Peak serum concentrations were observed within 4 hours post-administration in all groups, and MCP-1 concentrations tended to return to baseline within 48 hours. In the study group that received concurrent administration of CD3ε PepPOL,v2, a clear decrease in peak cytokine production was observed, with the greatest decrease occurring in the 250X combination group (approximately 98% decrease in 4 hours).

[0376] IL-6 Intravenous (IV) administration of 0.010 mg / kg CD20 TCE to male (n=1 / group) and female (n=1 / group) monkeys resulted in elevated serum IL-6 concentrations, with peak serum concentrations observed within 4 hours post-administration (8-12 times baseline). The response tended to return to baseline within 8-12 hours post-administration. Co-administration of 0.010 mg / kg CD20 with 0.016 mg / kg (25X) CD3ε PepPOL,v2 resulted in a moderate increase in IL-6 serum concentration in one of the two animals (n=1), which returned to baseline levels within 8-12 hours. In animals that received co-administration of 0.010 mg / kg CD20 TCE + 0.16 mg / kg (250X) CD3ε PepPOL,v2, no IL-6 stimulation was observed, suggesting that the pharmacodynamic effect of CD3ε PepPOL,v2 was saturated at this relative dose level.

[0377] TNFα, IFNγ, and IL-2 Intravenous (IV) administration of 0.010 mg / kg CD20 TCE elevated serum TNF-α levels in both animals, peaking 2–4 hours post-administration. An increase in IFN-γ levels was observed in one of the two animals 4 hours post-administration. All three cytokines returned to baseline by 8 hours post-administration. In one animal co-administered with 0.01 mg / kg CD20 TCE in combination with 0.016 mg / kg CD3ε PepPOL,v2, TNF-α and IFN-γ levels were not elevated (or not shown) at any time point, but this was not considered treatment-related. In both animals administered with CD20 TCE in combination with 0.160 mg / kg CD3ε PepPOL,v2, no TNF-α or IFN-γ levels exceeding assay LLOQ were detected at any time point. No detectable IL-2 response was observed in either group.

[0378] T lymphocyte activation When evaluated through both early (CD69+) and late (CD25+) markers monitored by flow cytometry, co-administration of CD3ε PepPOL,v2 had a significant effect on T cell activation. In general, the PD effect of co-administration of CD3ε PepPOL,v2, when observed over the duration of the 20-day study, reflected the effect of serum cytokines and clearly mitigated the dose response of CD3ε PepPOL,v2 to T cell activation compared to monotherapy with 0.010 mg / kg CD20 TCE.

[0379] clinical chemistry In response to CD20 TCE treatment, C-reactive protein (CRP) was monitored as a common marker of inflammation. Similar to the effect of CD3ε-PepPOL v2 on serum cytokines, a dose-dependent decrease in CRP was observed in the CD20 TCE / CD3ε-PepPOL v2 combination group compared to CD20 TCE monotherapy with 0.010 mg / kg intravenous administration.

[0380] CD20 TCE cytolytic activity Complete elimination of circulating CD20+ B lymphocytes was observed after intravenous (IV) administration of 0.010 mg / kg CD20 TCE alone or in combination with CD3ε-PepPOL v2. The apparent difference in CD20 TCE cytolytic activity was not distinguishable between the CD20 TCE control group administered IV at 0.010 mg / kg and the group administered CD3ε-PepPOL v2. This suggests that CD3ε-PepPOL v2 effectively decouples the cytokine / inflammatory response from the CD20 TCE PD effect.

[0381] Example 11: In silico mutation screening of conjugation peptides that bind to CD3ε domain I2C (SEQ ID NO: 26), I2E (SEQ ID NO: 382), and CD3 binders of heterodimer antibodies (SEQ ID NO: 381). A computer-based method was used to identify acceptable mutation combinations for the CD3ε peptide that binds to I2C. As described in Barlow et al. J.Phys.Chem (2018), the inventors used the Flex ddG protocol within the ROSETTA macromolecule modeling suit v.3.12 to computerically model and predict the change in binding free energy in mutations. This method generates a large association of the protein-peptide complex using a backrub protocol that samples the local scaffold and side-chain conformation around the mutation site. Next, the difference in binding free energy (△△G) between the wild-type and mutant complexes is calculated using Rosetta's Talaris energy function. A general additive model (GAM) is used to reweight Rosetta's predicted △△G against experimentally known values. △△G ≥ 1 indicates a destabilizing mutation, 1 < △△G ≤ -1 indicates a neutralizing mutation, and △△G < -1 indicates a stabilizing mutation. The inventors applied this protocol to a high-resolution crystal structure (1.35 Å) of the scFv region of I2C bound to the binding peptide (pE-DGNEELK) while maintaining the skeletal flexibility of the peptide chain. The inventors first performed a single mutation scan of the CD3ε peptide using mutation saturation. The inventors used a lower cutoff score of 1.2 for △△G when attempting to improve their sequence diversity in the expected binder pool. Next, based on single mutations with a △△G score of <1.2, the inventors applied double and quadruple combinatorial mutations at residues 2, 3, 4, and 6. Mutations of any amino acid from residue 5 onward were unacceptable, while mutagenesis from residue 1, Glu, to other residues was inconsistent with previous experimental data. Therefore, residues 1 and 5 were not mutated. Since residues 7 and 8 are not in the core binding pocket of I2C, they can be mutated to any amino acid. The inventors found that most of their predictions were consistent with combined experimental data from previous tests.

[0382] [Table 17]

[0383] A similar exercise was performed using I2E, a closely related variant of I2C with three different residues in the CDR. A high-resolution crystal structure (2.25 Å) of I2E scFv is available. First, the expected complex structure of I2E with the CD3ε peptide was constructed by superimposing the I2E crystal structure onto the I2C / CD3ε peptide cocrystal structure. Next, mutation saturation was performed at residues 2, 6, 7, and 8 using the Flex ddG protocol. From the expected results, two further mutations E and F were found to be acceptable at residue 2 compared to I2C. Mutations at residue 6 were not acceptable, while residues 7 and 8 could be substituted with any amino acid.

[0384] [Table 18]

[0385] High-resolution structures are not available for the CD3-binding domain of heterodimeric antibodies. Therefore, we created an scFv model using DeepAb, a state-of-the-art deep learning method that applies deep learning to predict the antibody Fv structure from the amino acid sequence. Next, to create a complex model with the CD3ε peptide, we aligned the predicted structure with I2C and performed a single mutation scan on residues 2-8. Peptide residues 2, 7, and 8 allow for any amino acid substitution, while residues 4 and 6 can be mutated for many other ami...

Claims

1. (i) A binding peptide that binds to an anti-CD3ε paratope of the CD3 epsilon (CD3ε) binding domain of TCE, wherein the anti-CD3ε paratope is for an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257, (ii) A linker covalently bonded to the C-terminus of the binding peptide, (iii) A half-life extension polymer covalently bonded to the linker, A T cell engager (TCE) masking molecule, including [specific component].

2. A TCE masking molecule according to claim 1, wherein the anti-CD3ε paratope binds to (i) at least one amino acid residue of K67 or S77 of SEQ ID NO: 257; or (ii) an epitope comprising at least amino acid residues K67, N68, I69, G70, S71, D72, E73, D74, H75, L76 and S77 of SEQ ID NO:

257.

3. A TCE masking molecule according to claim 1, wherein the anti-CD3ε paratope is for a CD3ε epitope consisting of the amino acid sequence of SEQ ID NO: 258 or the amino acid sequence of SEQ ID NO:

259.

4. A TCE masking molecule according to any one of claims 1 to 3, wherein the binding peptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids, preferably 5, 6, 7, 8, 9, or 10 amino acids, more preferably 10 amino acids.

5. A TCE masking molecule according to any one of claims 1 to 4, wherein the binding peptide is bound to an anti-CD3ε paratope and has at least the sequence X1X2X3X4EX5 (SEQ ID NO: 392) at its N-terminus (wherein X1 is Q, pyroglutamic acid (pE), or S; X2 is D, H, or N; X3 is G, F, or Y; X4 is N, E, S, T, V, or I; and X5 is E, L, P, or W).

6. A TCE masking molecule according to any one of claims 1 to 5, wherein the binding peptide is bound to an anti-CD3ε paratope and has at least the sequence X1DGX2EE (SEQ ID NO: 261) at its N-terminus (wherein X1 is Q, pE, or S, and X2 is N, E, S, T, V, or I).

7. A TCE masking molecule according to any one of claims 1 to 6, wherein the binding peptide is bound to an anti-CD3ε paratope and has at least the sequence X1X2X3X4EX5X6X7 (SEQ ID NO: 393) at its N-terminus (wherein X1 is Q, pyroglutamic acid (pE), or S; X2 is D, H, or N; X3 is G, F, or Y; X4 is N, E, S, T, V, or I; X5 is E, L, P, or W; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X7 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y).

8. A TCE masking molecule according to any one of claims 1 to 7, wherein the binding peptide is bound to an anti-CD3ε paratope and has at least the sequence pEX1X2X3EX4LK (SEQ ID NO: 396) at its N-terminus (wherein X1 is D, H, or N; X2 is G, F, or Y; X3 is N, E, S, T, V, or I; and X4 is E, L, P, or W).

9. A TCE masking molecule according to any one of claims 1 to 8, wherein the anti-CD3ε binding peptide comprises a palindrome.

10. A TCE masking molecule according to any one of claims 1 to 9, wherein the palindrome has K at a position between mirror-image amino acids.

11. A TCE masking molecule according to any one of claims 1 to 10, wherein the binding peptide is bound to an anti-CD3ε paratope and has at least one amino acid sequence of SEQ ID NOs: 263-284, 286-293, 295-303, 308-338, 385-388, 397-419, and 431 at its N-terminus.

12. A TCE masking molecule according to any one of claims 1 to 11, wherein the binding peptide is bound to an anti-CD3ε paratope and contains cysteine ​​at its C-terminus.

13. A TCE masking molecule according to any one of claims 1 to 12, wherein the binding peptide further comprises amino acids G and C or G and K at its C-terminus.

14. A TCE masking molecule according to any one of claims 1 to 8, wherein the binding peptide is bound to an anti-CD3ε paratope and has the name pEX1X2X3EX4X5X6GX7 (SEQ ID NO: 433) (where X1 is D, H or N, X2 is G, F or Y, preferably G or F, and X3 is N, H, Q, R, E, S, T, V, C, D, F, K A TCE masking molecule comprising the sequence L, M, W, Y or I, preferably N, E, H, Q or R, X4 is E, L, P or W, X5 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, X5 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, and X7 is C or K.

15. A TCE masking molecule according to any one of claims 1 to 8 and 14, wherein the binding peptide is bound to an anti-CD3ε paratope and contains the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (wherein X1 is D, H or N, X2 is G, F or Y, preferably G or F, X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y or I, preferably N, E, H, Q or R, most preferably N or E, and X4 is C or K).

16. A TCE masking molecule according to any one of claims 1 to 8 and 14 to 15, wherein the binding peptide is bound to an anti-CD3ε paratope and contains the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (wherein X1 is D, H or N, X2 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y or I, preferably E, N, H, Q or R, most preferably N or E, and X3 is C or K).

17. A TCE masking molecule according to any one of claims 1 to 8 and 14 to 16, wherein the binding peptide is bound to an anti-CD3ε paratope and contains the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (wherein X1 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E, and X2 is C or K).

18. A TCE masking molecule according to any one of claims 1 to 17, wherein the binding peptide is bound to an anti-CD3ε paratope and comprises the sequence of SEQ ID NOs: 297, 300, 303, 304, 305, 306, 307, 384, or 389.

19. A TCE masking molecule according to any one of claims 1 to 18, wherein the linker is (a) Disulfide linkers containing disulfide (R-S-S-R') (where R is a half-life extension polymer and R' is a binding peptide); (b) Free thiols containing linear or branched PEG, OPSS derivatives, maleimide, norbornene, acrylimide, acrylate, vinyl sulfone, or amine; or (c) Carboxylate linker containing an amine, amide, or epsilon-derivativeized acetyl bromide This is a TCE masking molecule.

20. A TCE masking molecule according to any one of claims 1 to 19, wherein the linker comprises the following portion: 【Chemistry 1】 Either one of the following is selected, or when the C-terminal amino acid of the binding peptide is K, the linker is of the formula 【Chemistry 2】 Maleimido-thiosuccinimide linker having; and When the C-terminal amino acid of the aforementioned binding peptide is C, the formula 【Transformation 3】 acetamido-thioether having Furthermore, when the C-terminal amino acid of the binding peptide is C, it is selected from disulfides having the formula R'-S-S-R, Here, R is a half-life extension polymer and R' is a binding peptide, forming a TCE masking molecule.

21. A TCE masking molecule according to any one of claims 1 to 20, wherein the half-life extending polymer is mono-methoxypolyethylene glycol (mPEG), linear, 2-arm, 4-arm, 8-arm polyethylene glycol (PEG); PLGA; peptide acrylate, polyglycerol, polyoxazoline, polyvinylpyrrolidone, polyacrylamide, poly(N-acryloylmorpholine), poly(N,N-dimethylacrylamide), pre(ply)(2-hydroxypropyl methacrylamide), polysarcosine, poly(2-hydroxyethyl methacrylamide), hyaluronic acid, sialic acid, poly[(organo)phosphazene] or heparin.

22. A TCE masking molecule according to any one of claims 1 to 21, wherein the half-life extension polymer is 【Chemistry 4】 (wherein n is an integer of about 20 to 200, preferably 20 to 169, more preferably 20, 103 or 113) 【Transformation 5】 (wherein n is an integer between approximately 40 and 400, preferably approximately 40 or approximately 208) or, 【Transformation 6】 A TCE masking molecule (wherein n is equal to a number of about 80 to about 700, preferably about 80 to about 675, more preferably about 83, about 166 or about 417).

23. A TCE masking molecule according to any one of claims 1 to 22, wherein the half-life extension polymer is an unbranched linear or branched 2-arm, 4-arm or 8-arm PEG, preferably linear or 4-arm, having a molecular weight of about 2 kDa to about 60 kDa, preferably about 4 kDa to about 30 kDa or 10 kDa to about 30 kDa or more preferably about 5 or about 20 kDa.

24. A TCE masking molecule according to any one of claims 1 to 23, wherein the half-life extension polymer is a branched polymer, preferably a 2-arm, 4-arm, or 8-arm PEG having a molecular weight of about 2 kDa to about 60 kDa, wherein a plurality of binding peptides are each linked via linkers, preferably 2, 4, or 8 binding peptides are linked to one half-life extension polymer.

25. A TCE masking molecule according to any one of claims 1 to 8, 14 to 17, and 19 to 24, comprising the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (wherein X1 is D, H, or N; X2 is G, F, or Y, preferably G or F; and X3 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E), (a.) Here, if X4 is C; formula 【Transformation 7】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Transformation 8】 (b) a half-life extension polymer which is PEG, and (b) if X4 is K; formula 【Chemistry 9】 An acetamide-thioether having the following formula, and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. 【Chemistry 10】 A PEG half-life extension polymer, comprising TCE masking molecule.

26. A TCE masking molecule according to any one of claims 1 to 8, 14 to 17, and 19 to 25, comprising the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (wherein X1 is D, H, or N, and X2 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably E, N, H, Q, or R, most preferably N or E), (a.) When X3 is C; formula 【Chemistry 11】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 12】 (b) a half-life extension polymer which is PEG, and (b) if X3 is K; formula 【Chemistry 13】 An acetamide-thioether having; and a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 14】 A PEG half-life extension polymer, comprising TCE masking molecule.

27. A TCE masking molecule according to any one of claims 1 to 8, 14 to 17, and 19 to 26, comprising the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is N or E), and (a) when X2 is C, formula 【Chemistry 15】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 kDa or about 20 kDa, the following formula 【Chemistry 16】 (b) a half-life extension polymer which is PEG, and (b) a sequence of pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is N or E) and X2 is K; formula 【Chemistry 17】 An acetamide-thioether having; and a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [Chemistry 18] A PEG half-life extension polymer, comprising TCE masking molecule.

28. A TCE masking molecule according to any one of claims 1 to 8, 14 to 17, and 19 to 27, (a) The amino acid sequence of Sequence ID No. 304, 306, 384 or 389, formula 【Chemistry 19】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula 【Chemistry 20】 A PEG containing a half-life extension polymer, or (b) The amino acid sequence of Sequence ID No. 305 or 307, formula 【Chemistry 21】 An acetamido-thioether having, The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula 【Chemistry 22】 The PEG half-life extension polymer, A TCE masking molecule containing this molecule.

29. A TCE masking molecule according to any one of claims 1 to 28, comprising any combination (a.) to (k.) of a binding peptide, a linker, and a half-life extending PEG polymer (where R' represents the binding peptide and R represents the half-life extending PEG polymer): Table 1 Table 2 Table 3

30. A TCE masking molecule according to any one of claims 1 to 29, wherein the half-life is about 1 hour to about 48 hours, preferably about 1.5 hours to about 24 hours, more preferably about 1.5 hours to about 4 hours, or more preferably about 2 hours.

31. A method for reducing the severity of cytokine release syndrome in human subjects receiving TCE treatment, comprising administering an effective dose of a T cell engager masking molecule to the human subject, wherein the TCE masking molecule is (i.) A binding peptide that binds to an anti-CD3ε paratope of the CD3ε binding domain of TCE, wherein the anti-CD3ε paratope is for an epitope located within CD3ε, and CD3ε comprises the amino acid sequence of SEQ ID NO: 257; (ii.) A linker covalently bonded to the C-terminus of the binding peptide; (iii.) At least one half-life extension polymer, preferably with a half-life of at least 2 kDa, covalently bonded to the linker, Methods that include...

32. The method according to claim 31, wherein the T cell engager masking molecule is The compound peptide contains a conjugated peptide that binds to the anti-CD3ε paratope and comprises the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (where X1 is D, H, or N; X2 is G, F, or Y, preferably G or F; and X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R), (a.) when X4 is C; formula 【Chemistry 23】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 24】 (b) a half-life extension polymer which is PEG, and (b) if X4 is K; formula 【Chemistry 25】 An acetamido-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 26】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

33. The method according to claim 31 or 32, wherein the T cell engager masking molecule is The anti-CD3ε paratope contains a conjugated peptide comprising the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (where X1 is D, H, or N, and X2 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, H, Q, or R), and (a.) when X3 is C, the formula 【Chemistry 27】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 28】 (b) a half-life extension polymer which is PEG, and (b) if X3 is K; formula 【Chemistry 29】 An acetamido-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Transformation 30】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

34. A method according to any one of claims 31 to 33, wherein the T cell engager masking molecule is The anti-CD3ε paratope contains a conjugated peptide comprising the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is D, H, or N, and X1 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E), and (a.) when X2 is C; formula 【Chemistry 31】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 32】 (b) a half-life extension polymer which is PEG, and (b) if X2 is K; formula 【Transformation 33】 An acetamido-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Transformation 34】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

35. A method according to any one of claims 31 to 34, wherein the T cell engager masking molecule is (a) The amino acid sequences of SEQ ID NOs: 304, 306, 384 or 389, and formula 【Chemistry 35】 A linker selected from maleimido-thiosuccinimide linkers having the formula R'-S-S-R and disulfides having the formula R'-S-S-R; And, the following formula, having a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. 【Transformation 36】 A PEG half-life extension polymer; or (b) The amino acid sequence of SEQ ID NO: 305 or 307; formula 【Chemistry 37】 acetamido-thioether having And, the following formula, having a molecular weight of about 5 kDa to about 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. 【Chemistry 38】 A PEG half-life extended polymer (where R' represents the binding peptide and R represents the half-life extended PEG polymer) A method comprising, or preferably comprising, the following.

36. The method according to any one of claims 31 to 35, wherein the immunotherapy comprises administering a T-cell engager.

37. The method according to any one of claims 31 to 36, wherein the molar ratio of the TCE masking molecule to the T cell engager is in the range of about 1000 to about 1, or about 250:1 to about 10:1, or about 100:1 to about 25:

1.

38. The method according to any one of claims 31 to 37, wherein the TCE masking molecule is administered before, during, or after administration of TCE.

39. A method according to any one of claims 31 to 38, wherein the TCE is (i) A TCE comprising at least first, second, and third domains in order from amino to carboxyl, The first domain binds to a target cell surface antigen, and the antigen is preferably a tumor antigen; The second domain binds to the CD3ε epitope; The third domain comprises two polypeptide monomers, each containing a hinge, a CH2 domain, and a CH3 domain, the two polypeptide monomers fused to each other via a peptide linker, and the third domain comprises, in order from amino to carboxyl: hinge-CH2-CH3-linker-hinge-CH2-CH3, TCE; (ii) A first binding domain comprising scFv, scFab, or Fab that binds to a first target cell surface antigen (TAA1); A second binding domain comprising scFv, scFab, or Fab that binds to an extracellular epitope on the human CD3ε chain; Spacers that are crystallizable from single-chain fragments (scFc), human serum albumin (HSA), programmed death receptor 1 (PD1), or hetero-fragment crystallizable (heteroFC); A third binding domain comprising scFv, scFab, or Fab that binds to a second target cell surface antigen (TAA2); and A fourth binding domain comprising scFv, scFab, or Fab that binds to the CD3ε epitope. A TCE that includes, The first binding domain binds to the first target cell surface antigen, and the third binding domain simultaneously binds to the second target cell surface antigen, and the first target cell surface antigen and the second target cell surface antigen are on the same target cell. Preferably a single polypeptide chain, The first target cell surface antigen and the second target cell surface antigen are not the same, The first binding domain and the second binding domain form a first dual specificity entity, the third binding domain and the fourth binding domain form a second dual specificity entity, and The spacer entity is located between the first bisingular entity and the second bisingular entity. TCE, (iii) IgG-based bispecific antibody; or (iv) heterodimer antibody The method.

40. A method according to claim 39, wherein the TCE under claim 39(i) is a single-chain molecule.

41. The method according to claim 39 or 40, wherein the glycosylation site at the Kabat position 314 of the CH2 domain in the third domain of the TCE under (i) and (ii) of claim 39 is removed by N314X substitution, where X is any amino acid other than Q.

42. A method according to any one of claims 39 to 41, wherein each of the polypeptide monomers of the third domain contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 437 to 444, or contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 437 to 444.

43. The method according to any one of claims 39 to 42, wherein the CH2 domain includes an intradomain cysteine ​​disulfide bridge.

44. A method according to any one of claims 39 to 43, wherein the tumor antigen is CDH19, CDH3, MSLN, DLL3, FLT3, EGFRvIII, BCMA, PSMA, CD33, CD19, CD20, CLDN18.2, CLDN6, MUC17, EpCAM, STEAP1, or CD70.

45. A method according to any one of claims 39 to 44, wherein the TCE under claim 39(i) is sequentially in the direction from amino to carboxyl, (a) The first domain; (b) A peptide linker having any one amino acid sequence of SEQ ID NOs: 187-189; (c) Second domain; (d) A peptide linker having any one amino acid sequence of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) A first peptide monomer of the third domain having the amino acid sequence of any of SEQ ID NOs: 437 to 444; (f) A peptide linker having any one amino acid sequence of SEQ ID NOs: 191, 192, 193, and 194; and (g) A second peptide monomer of the third domain having the amino acid sequence of any of SEQ ID NOs: 437-444 Methods that include...

46. A method according to any one of claims 39 to 45, wherein the first binding domain and / or third binding domain of the TCE under claim 39 (ii) is (a) CDR-H1 as shown in Sequence ID No. 4, CDR-H2 as shown in Sequence ID No. 5, CDR-H3 as shown in Sequence ID No. 6, CDR-L1 as shown in Sequence ID No. 1, CDR-L2 as shown in Sequence ID No. 2, and CDR-L3 as shown in Sequence ID No. 3; (b) CDR-H1 as shown in Sequence ID 29, CDR-H2 as shown in Sequence ID 30, CDR-H3 as shown in Sequence ID 31, CDR-L1 as shown in Sequence ID 34, CDR-L2 as shown in Sequence ID 35, and CDR-L3 as shown in Sequence ID 36; (c) CDR-H1 as shown in sequence number 42, CDR-H2 as shown in sequence number 43, CDR-H3 as shown in sequence number 44, CDR-L1 as shown in sequence number 45, CDR-L2 as shown in sequence number 46, and CDR-L3 as shown in sequence number 47; (d) CDR-H1 as shown in Sequence ID 53, CDR-H2 as shown in Sequence ID 54, CDR-H3 as shown in Sequence ID 55, CDR-L1 as shown in Sequence ID 56, CDR-L2 as shown in Sequence ID 57, and CDR-L3 as shown in Sequence ID 58; (e) CDR-H1 as shown in sequence number 65, CDR-H2 as shown in sequence number 66, CDR-H3 as shown in sequence number 67, CDR-L1 as shown in sequence number 68, CDR-L2 as shown in sequence number 69, and CDR-L3 as shown in sequence number 70; (f) CDR-H1 as shown in sequence number 83, CDR-H2 as shown in sequence number 84, CDR-H3 as shown in sequence number 85, CDR-L1 as shown in sequence number 86, CDR-L2 as shown in sequence number 87, and CDR-L3 as shown in sequence number 88; (g) CDR-H1 as shown in SEQ ID NO: 94, CDR-H2 as shown in SEQ ID NO: 95, CDR-H3 as shown in SEQ ID NO: 96, CDR-L1 as shown in SEQ ID NO: 97, CDR-L2 as shown in SEQ ID NO: 98, and CDR-L3 as shown in SEQ ID NO: 99; (h) CDR-H1 as shown in sequence number 105, CDR-H2 as shown in sequence number 106, CDR-H3 as shown in sequence number 107, CDR-L1 as shown in sequence number 109, CDR-L2 as shown in sequence number 110, and CDR-L3 as shown in sequence number 111; (i) CDR-H1 as shown in sequence number 115, CDR-H2 as shown in sequence number 116, CDR-H3 as shown in sequence number 117, CDR-L1 as shown in sequence number 118, CDR-L2 as shown in sequence number 119, and CDR-L3 as shown in sequence number 120; (j) CDR-H1 as shown in sequence number 126, CDR-H2 as shown in sequence number 127, CDR-H3 as shown in sequence number 128, CDR-L1 as shown in sequence number 129, CDR-L2 as shown in sequence number 130, and CDR-L3 as shown in sequence number 131; (k) CDR-H1 as shown in sequence number 137, CDR-H2 as shown in sequence number 138, CDR-H3 as shown in sequence number 139, CDR-L1 as shown in sequence number 140, CDR-L2 as shown in sequence number 141, and CDR-L3 as shown in sequence number 142; (l) CDR-H1 as shown in sequence number 152, CDR-H2 as shown in sequence number 153, CDR-H3 as shown in sequence number 154, CDR-L1 as shown in sequence number 155, CDR-L2 as shown in sequence number 156, and CDR-L3 as shown in sequence number 157; (m) CDR-H1 as shown in sequence number 167, CDR-H2 as shown in sequence number 168, CDR-H3 as shown in sequence number 169, CDR-L1 as shown in sequence number 170, CDR-L2 as shown in sequence number 171, and CDR-L3 as shown in sequence number 172; (n) CDR-H1 as shown in sequence number 203, CDR-H2 as shown in sequence number 204, CDR-H3 as shown in sequence number 205, CDR-L1 as shown in sequence number 206, CDR-L2 as shown in sequence number 207, and CDR-L3 as shown in sequence number 208; (o) CDR-H1 as shown in sequence number 214, CDR-H2 as shown in sequence number 215, CDR-H3 as shown in sequence number 216, CDR-L1 as shown in sequence number 217, CDR-L2 as shown in sequence number 218, and CDR-L3 as shown in sequence number 219; (p) CDR-H1 as shown in sequence number 226, CDR-H2 as shown in sequence number 227, CDR-H3 as shown in sequence number 228, CDR-L1 as shown in sequence number 229, CDR-L2 as shown in sequence number 230, and CDR-L3 as shown in sequence number 231; (q) CDR-H1 as shown in Sequence ID No. 238, CDR-H2 as shown in Sequence ID No. 239, CDR-H3 as shown in Sequence ID No. 240, CDR-L1 as shown in Sequence ID No. 241, CDR-L2 as shown in Sequence ID No. 242, and CDR-L3 as shown in Sequence ID No. 243; and (r) CDR-H1 as shown in sequence number 420, CDR-H2 as shown in sequence number 421, CDR-H3 as shown in sequence number 422, CDR-L1 as shown in sequence number 423, CDR-L2 as shown in sequence number 424, and CDR-L3 as shown in sequence number 425 A method comprising a VH region including CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of the above, and a VL region including CDR-L1, CDR-L2, and CDR-L3.

47. A method according to any one of claims 39 to 46, wherein the TCE comprises the amino acid sequence of SEQ ID NOs: 17, 52, 63, 81, 93, 104, 114, 125, 136, 147, 162, 177, 213, 226, 238, 248, 255 or 430, preferably 104 or 255.

48. The method according to claim 39, wherein the heterodimer antibody is a) (i) First variable heavy chain domain; (ii) A first steady heavy chain comprising a first CH1 domain and a first Fc domain; (iii) an scFv bound to CD3ε, comprising an scFv variable light chain domain, an scFv linker and an scFv variable heavy chain domain. Includes, The scFv is covalently bonded between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain using a domain linker. A first monomer containing the first heavy chain; (b) A second monomer comprising a second heavy chain comprising a second variable heavy chain domain and a second steady heavy chain comprising a second Fc domain; (c) A common light chain including a variable light chain domain and a constant light chain domain (wherein the first variable heavy chain domain and the variable light chain domain bind to human STEAP1), Includes, The second variable chain domain and the variable light chain domain bind to human STEAP1, where, (1) The first monomer contains the amino acid sequence of SEQ ID NO: 375, the second monomer contains the sequence of SEQ ID NO: 374, and the common light chain contains the sequence of SEQ ID NO: 373; (2) The first monomer contains the sequence of SEQ ID NO: 380, the second monomer contains the sequence of SEQ ID NO: 376, and the common light chain contains the sequence of SEQ ID NO: 373; or (3) The first monomer contains the sequence of SEQ ID NO: 379, the second monomer contains the sequence of SEQ ID NO: 378, and the common light chain contains the sequence of SEQ ID NO: 377, method.

49. The TCE masking molecule or method according to any one of claims 1 to 48, wherein the CD3ε-binding domain has 80, 90, 95, or 99% sequence identity with the sequence of SEQ ID NO: 26, 381, 382, ​​or 383, or has the sequence of SEQ ID NO: 26, 381, 382, ​​or 383.

50. A TCE masking molecule for use in reducing the severity of cytokine release in human subjects undergoing TCE treatment, wherein the TCE masking molecule is (i.) A binding peptide that binds to an anti-CD3ε paratope of the CD3ε binding domain of TCE, wherein the anti-CD3ε paratope is for an epitope located within CD3ε, and CD3ε comprises the amino acid sequence of SEQ ID NO: 257; (ii) a linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life extension polymer covalently bonded to the linker, A TCE masking molecule containing this molecule.

51. A method for reducing in vivo exposure to TCE after TCE administration, wherein the administration is preferably via the i.v. or s.c. route. (a) (i.) A binding peptide that binds to an anti-CD3ε paratope of the CD3ε binding domain of TCE, wherein the anti-CD3ε paratope is an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257, (ii) a linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life-extending polymer covalently bonded to the linker, wherein the binding peptide binds to the anti-CD3ε paratope at a Kd at least 1.5 times lower, preferably at least 2 times lower, more preferably 3 times lower, than the Kd of the amino acid sequence of SEQ ID NO: 262 that binds to the anti-CD3ε paratope. We provide a TCE masking molecule. (b) The step of administering the TCE masking molecule in a molar excess of about 10 to about 1000 or about 25 to about 250 with respect to the TCE. Methods that include...

52. The method according to claim 51, wherein the TCE masking molecule is administered before, during, or after administration of the TCE.

53. The method according to claim 51 or 52, wherein the T cell engager masking molecule is The compound peptide contains a conjugated peptide that binds to the anti-CD3ε paratope and comprises the sequence pEX1X2X3EELKGX4 (SEQ ID NO: 434) (where X1 is D, H, or N; X2 is G, F, or Y, preferably G or F; and X3 is N, H, Q, R, E, S, T, V, C, D, F, KL, M, W, Y, or I, preferably N, E, H, Q, or R), (a.) when X4 is C; formula 【Chemistry 39】 A maleimide-thiosuccinimide linker having the formula R'-S-S-R, and a linker selected from disulfides having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 40】 (b) a half-life extension polymer which is PEG, and (b) if X4 is K; formula 【Chemistry 41】 An acetamide-thioether having the following formula, and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa. 【Chemistry 42】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

54. A method according to any one of claims 51 to 53, wherein the T cell engager masking molecule is The anti-CD3ε paratope contains a conjugated peptide comprising the sequence pEX1GX2EELKGX3 (SEQ ID NO: 435) (where X1 is D, H, or N, and X2 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, H, Q, or R), (a.) when X3 is C; formula 【Chemistry 43】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 44】 (b) a half-life extension polymer which is PEG, and (b) if X3 is K; formula 【Chemistry 45】 An acetamido-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 46】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

55. A method according to any one of claims 51 to 54, wherein the T cell engager masking molecule is The anti-CD3ε paratope contains a conjugated peptide comprising the sequence pEDGX1EELKGX2 (SEQ ID NO: 436) (where X1 is D, H, or N, and X1 is N, H, Q, R, E, S, T, V, C, D, F, K, L, M, W, Y, or I, preferably N, E, H, Q, or R, most preferably N or E), and (a.) when X2 is C; formula 【Chemistry 47】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; and a linker having a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula 【Chemistry 48】 (b) a half-life extension polymer which is PEG, and (b) if X2 is K; formula 【Chemistry 49】 An acetamido-thioether having; and a molecular weight of about 5 to 20 kDa, preferably about 5 kDa, about 10 kDa, or about 20 kDa, the following formula [Transformation 50] A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

56. A method according to any one of claims 51 to 55, wherein the T cell engager masking molecule is (a) The amino acid sequence of Sequence ID No. 304, 306, 384 or 389, formula 【Chemistry 51】 A linker selected from a maleimide-thiosuccinimide linker having the formula R'-S-S-R and a disulfide having the formula R'-S-S-R; The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula 【Chemistry 52】 A PEG containing a half-life extension polymer; or, (b) The amino acid sequence of Sequence ID No. 305 or 307, formula 【Chemistry 53】 An acetamido-thioether having, The molecular weight is approximately 5 kDa to approximately 20 kDa, preferably approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa, and the following formula 【Chemistry 54】 A PEG half-life extension polymer and (where R' represents the binding peptide and R represents the half-life extension PEG polymer) method.

57. The method according to any one of claims 51 to 56, wherein the immunotherapy comprises administering a T cell engager.

58. The method according to any one of claims 51 to 57, wherein the molar ratio of the TCE masking molecule to the TCE is in the range of about 1000 to about 1, or about 250:1 to about 10:1, or about 100:1 to about 25:

1.

59. The method according to any one of claims 51 to 58, wherein the TCE masking molecule is administered before, during, or after administration of TCE.

60. A method according to any one of claims 51 to 58, wherein the TCE is (i) A TCE comprising at least first, second, and third domains in order from amino to carboxyl, The first domain binds to a target cell surface antigen, which is preferably a tumor antigen; The second domain binds to the epitope of CD3ε; The third domain comprises two polypeptide monomers, each containing a hinge, a CH2 domain, and a CH3 domain, the two polypeptide monomers fused to each other via a peptide linker, and the third domain comprises, in order from amino to carboxyl: hinge-CH2-CH3-linker-hinge-CH2-CH3, TCE; (ii) A first binding domain comprising scFv, scFab, or Fab, which binds to a first target cell surface antigen (TAA1); A second binding domain comprising scFv, scFab, or Fab, which binds to an extracellular epitope on the human CD3ε chain; A spacer that is crystallizable as a single-chain fragment (scFc), human serum albumin (HSA), programmed death receptor 1 (PD1), or heterofragment (heteroFC), A third binding domain comprising scFv, scFab, or Fab, which binds to a second target cell surface antigen (TAA2); A fourth binding domain comprising scFv, scFab, or Fab that binds to the CD3ε epitope, A TCE that includes; The first binding domain binds to the first target cell surface antigen, and the third binding domain simultaneously binds to the second target cell surface antigen, and the first target cell surface antigen and the second target cell surface antigen are on the same target cell. The TCE is preferably a single polypeptide chain; The first target cell surface antigen and the second target cell surface antigen are not the same, The first binding domain and the second binding domain form a first dual specificity entity, the third binding domain and the fourth binding domain form a second dual specificity entity, and Here, The spacer entity is located between the first bispecific entity and the second bispecific entity in a TCE; (iii) IgG-based bispecific antibody; or (iv) Heterodimeric antibody The method.

61. The method according to claim 60, wherein the TCE(i.) described in claim 60 is a single-chain molecule.

62. The method according to claim 60 or 61, wherein the glycosylation site at the Kabat position 314 of the CH2 domain in the third domain of the TCE is removed by N314X substitution, where X is any amino acid except Q.

63. The method according to any one of claims 60 to 62, wherein each polypeptide monomer of the third domain contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 437 to 444, or contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 437 to 444.

64. The method according to any one of claims 60 to 63, wherein the CH2 domain includes an intradomain cysteine ​​disulfide bridge.

65. The method according to any one of claims 60 to 64, wherein the tumor antigen is CDH19, CDH3, MSLN, DLL3, FLT3, EGFRvIII, BCMA, PSMA, CD33, CD19, CD20, CLDN18.2, CLDN6, MUC17, EpCAM, STEAP1, or CD70.

66. A method according to any one of claims 60 to 65, wherein the TCE below (i) in claim 59 is sequentially from amino to carboxyl, (a) The first domain; (b) A peptide linker having any one amino acid sequence of SEQ ID NOs: 187-189; c. Second domain; (d) A peptide linker having any one amino acid sequence of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) A first peptide monomer of the third domain having the amino acid sequence of any of SEQ ID NOs: 437 to 444; (f) A peptide linker having any one amino acid sequence of SEQ ID NOs: 191, 192, 193, and 194; and (g) A second peptide monomer of the third domain having the amino acid sequence of any of SEQ ID NOs: 437-444 Methods that include...

67. A method according to any one of claims 60 to 66, wherein the first and / or third binding domain of the TCE under claim 39(ii) is (a) CDR-H1 as shown in Sequence ID No. 4, CDR-H2 as shown in Sequence ID No. 5, CDR-H3 as shown in Sequence ID No. 6, CDR-L1 as shown in Sequence ID No. 1, CDR-L2 as shown in Sequence ID No. 2, and CDR-L3 as shown in Sequence ID No. 3; (b) CDR-H1 as shown in Sequence ID 29, CDR-H2 as shown in Sequence ID 30, CDR-H3 as shown in Sequence ID 31, CDR-L1 as shown in Sequence ID 34, CDR-L2 as shown in Sequence ID 35, and CDR-L3 as shown in Sequence ID 36; (c) CDR-H1 as shown in sequence number 42, CDR-H2 as shown in sequence number 43, CDR-H3 as shown in sequence number 44, CDR-L1 as shown in sequence number 45, CDR-L2 as shown in sequence number 46, and CDR-L3 as shown in sequence number 47; (d) CDR-H1 as shown in sequence number 53, CDR-H2 as shown in sequence number 54, CDR-H3 as shown in sequence number 55, CDR-L1 as shown in sequence number 56, CDR-L2 as shown in sequence number 57, and CDR-L3 as shown in sequence number 58, (e) CDR-H1 as shown in sequence number 65, CDR-H2 as shown in sequence number 66, CDR-H3 as shown in sequence number 67, CDR-L1 as shown in sequence number 68, CDR-L2 as shown in sequence number 69, and CDR-L3 as shown in sequence number 70, (f) CDR-H1 as shown in sequence number 83, CDR-H2 as shown in sequence number 84, CDR-H3 as shown in sequence number 85, CDR-L1 as shown in sequence number 86, CDR-L2 as shown in sequence number 87, and CDR-L3 as shown in sequence number 88, (g) CDR-H1 as shown in sequence number 94, CDR-H2 as shown in sequence number 95, CDR-H3 as shown in sequence number 96, CDR-L1 as shown in sequence number 97, CDR-L2 as shown in sequence number 98, and CDR-L3 as shown in sequence number 99, (h) CDR-H1 as shown in sequence number 105, CDR-H2 as shown in sequence number 106, CDR-H3 as shown in sequence number 107, CDR-L1 as shown in sequence number 109, CDR-L2 as shown in sequence number 110, and CDR-L3 as shown in sequence number 111, (i) CDR-H1 as shown in sequence number 115, CDR-H2 as shown in sequence number 116, CDR-H3 as shown in sequence number 117, CDR-L1 as shown in sequence number 118, CDR-L2 as shown in sequence number 119, and CDR-L3 as shown in sequence number 120, (j) CDR-H1 as shown in sequence number 126, CDR-H2 as shown in sequence number 127, CDR-H3 as shown in sequence number 128, CDR-L1 as shown in sequence number 129, CDR-L2 as shown in sequence number 130, and CDR-L3 as shown in sequence number 131, (k) CDR-H1 as shown in sequence number 137, CDR-H2 as shown in sequence number 138, CDR-H3 as shown in sequence number 139, CDR-L1 as shown in sequence number 140, CDR-L2 as shown in sequence number 141, and CDR-L3 as shown in sequence number 142, (l) CDR-H1 as shown in sequence number 152, CDR-H2 as shown in sequence number 153, CDR-H3 as shown in sequence number 154, CDR-L1 as shown in sequence number 155, CDR-L2 as shown in sequence number 156, and CDR-L3 as shown in sequence number 157, (m) CDR-H1 as shown in sequence number 167, CDR-H2 as shown in sequence number 168, CDR-H3 as shown in sequence number 169, CDR-L1 as shown in sequence number 170, CDR-L2 as shown in sequence number 171, and CDR-L3 as shown in sequence number 172, (n) CDR-H1 as shown in sequence number 203, CDR-H2 as shown in sequence number 204, CDR-H3 as shown in sequence number 205, CDR-L1 as shown in sequence number 206, CDR-L2 as shown in sequence number 207, and CDR-L3 as shown in sequence number 208, (o) CDR-H1 as shown in sequence number 214, CDR-H2 as shown in sequence number 215, CDR-H3 as shown in sequence number 216, CDR-L1 as shown in sequence number 217, CDR-L2 as shown in sequence number 218, and CDR-L3 as shown in sequence number 219, (p) CDR-H1 as shown in sequence number 226, CDR-H2 as shown in sequence number 227, CDR-H3 as shown in sequence number 228, CDR-L1 as shown in sequence number 229, CDR-L2 as shown in sequence number 230, and CDR-L3 as shown in sequence number 231; (q) CDR-H1 as shown in Sequence ID No. 238, CDR-H2 as shown in Sequence ID No. 239, CDR-H3 as shown in Sequence ID No. 240, CDR-L1 as shown in Sequence ID No. 241, CDR-L2 as shown in Sequence ID No. 242, and CDR-L3 as shown in Sequence ID No. 243; and (r) CDR-H1 as shown in sequence number 420, CDR-H2 as shown in sequence number 421, CDR-H3 as shown in sequence number 422, CDR-L1 as shown in sequence number 423, CDR-L2 as shown in sequence number 424, and CDR-L3 as shown in sequence number 425 A method comprising a VH region including CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of the above, and a VL region including CDR-L1, CDR-L2, and CDR-L3.

68. A method according to any one of claims 60 to 67, wherein the TCE comprises the amino acid sequence of SEQ ID NOs: 17, 52, 63, 81, 93, 104, 114, 125, 136, 147, 162, 177, 213, 226, 238, 248, 255 or 430, preferably 104 or 255.

69. The method according to claim 60, wherein the heterodimer antibody is (a) (i) First variable heavy chain domain; (ii) A first steady heavy chain comprising a first CH1 domain and a first Fc domain; (iii) an scFv bound to CD3ε, comprising an scFv variable light chain domain, an scFv linker and an scFv variable heavy chain domain. It includes a first heavy chain containing, The scFv is covalently bonded between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain using a domain linker. The first monomer and, (b) comprising a second variable heavy chain domain and a second constant heavy chain including a second Fc domain A second monomer containing a second heavy chain, (c) A common light chain comprising a variable light chain domain and a constant light chain domain, wherein the first variable heavy chain domain and the variable light chain domain bind to human STEAP1, Includes, The second variable heavy chain domain and the variable light chain domain bind to human STEAP1. (1) The first monomer contains the amino acid sequence of SEQ ID NO: 375, the second monomer contains the sequence of SEQ ID NO: 374, and the common light chain contains the sequence of SEQ ID NO: 373; (2) The first monomer contains the sequence of SEQ ID NO: 380, the second monomer contains the sequence of SEQ ID NO: 376, and the common light chain contains the sequence of SEQ ID NO: 373; or (3) The first monomer contains the sequence of SEQ ID NO: 379, the second monomer contains the sequence of SEQ ID NO: 378, and the common light chain contains the sequence of SEQ ID NO: 377, method.

70. A TCE masking molecule or method according to any one of claims 1 to 69, wherein the CD3ε-binding domain has 80, 90, 95, or 99% sequence identity with the sequence of SEQ ID NO: 26, 381, 382, ​​or 383, or has the sequence of SEQ ID NO: 26, 381, 382, ​​or 383.

71. A TCE masking molecule for use in reducing in vivo exposure to TCE after administration of the TCE molecule, wherein the administration is preferably via the i.v. or s.c. route, and the method is (a) (i.) A binding peptide that binds to an anti-CD3ε paratope of the CD3ε binding domain of TCE, wherein the anti-CD3ε paratope is an epitope located within CD3ε, and CD3ε contains the amino acid sequence of SEQ ID NO: 257, (ii) a linker covalently bonded to the C-terminus of the binding peptide; (iii) A half-life extension polymer covalently bonded to the linker, Includes, The binding peptide binds to the anti-CD3ε paratope at a Kd that is at least 1.5 times lower, preferably at least 2 times lower, more preferably 3 times lower, than the Kd of the amino acid sequence of SEQ ID NO: 262 that binds to the anti-CD3ε paratope. We provide a TCE masking molecule. (b) The step of administering the TCE masking molecule before, during, or after administration of the TCE in a molar excess of about 10 to about 1000 or about 25 to about 250 with respect to the TCE. A TCE masking molecule containing this molecule.

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