Perfusion control based on automated biomass in production of biopharmaceutical

Abstract

To provide an adapted perfusion or a continuous perfusion production process which includes a controlled perfusion amount based on an automated biomass, and secures a more efficient process.SOLUTION: A process includes a first control loop for measuring and regulating a culture medium level in a bioreactor, and a second control loop including a dielectric constant probe or a Raman probe for measuring and regulating a biomass in the bioreactor. The first control loop and the second control loop are integrated by an integration unit.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to biotechnology methods, in particular to automated aspects of (preferably continuous) manufacturing processes for the production of biopharmaceuticals such as antibodies. [Background technology]

[0002] Despite advances in manufacturing, new biologics (protein-based medicines) require new, optimized manufacturing processes to avoid negative effects on product quality, such as protein aggregation, which impacts upstream manufacturing, downstream production, storage, and application.

[0003] Such novel protein-based pharmaceuticals include antibodies, e.g., bispecific and / or monoclonal antibodies. Bispecific antibodies are artificial proteins that can simultaneously bind to two different types of antigens. They are known in several structural forms and are currently being explored for applications in cancer immunotherapy and drug delivery (Fan, Gaowei; Wang, Zujian; Hao, Mingju; Li, Jinming (2015). "Bispecific antibodies and their applications." Journal of Hematology & Oncology. 8:130).

[0004] In general, bispecific antibodies may be IgG-like, i.e., full-length bispecific antibodies, or non-IgG-like bispecific antibodies that are not full-length antibody constructs. Full-length bispecific antibodies typically retain the conventional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab regions bind to different antigens. Non-full-length bispecific antibodies completely lack the Fc region. These include chemically linked Fabs consisting of only the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFvs). There are also fusion proteins that mimic the variable domains of two antibodies. The most advanced of these new formats is the BiTE® bispecific T cell engager molecule (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). "Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies". International Journal of Molecular Sciences. 18(1):48).

[0005] Bispecific molecules, such as BiTE® molecules, are recombinant protein constructs composed of two flexibly linked antibody-derived binding domains. One binding domain of a BiTE® molecule is specific for a selected tumor-associated surface antigen on target cells; the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. Due to their special design, BiTE® antibody constructs are uniquely suited to transiently link T cells to target cells while simultaneously potently activating the intrinsic cytolytic capacity of T cells against target cells. Important further developments of the first-generation BiTE® molecules deployed in the clinic as AMG 103 and AMG 110 (see WO 99 / 54440 and WO 2005 / 040220) provided bispecific molecules that bind to a context-independent epitope at the N-terminus of the CD3 epsilon chain (WO 2008 / 119567). BiTE® molecules that bind to this selected epitope not only lack cross-species specificity for human and marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), or squirrel monkey (Saimiri sciureus) CD3ε chains, but also, because they recognize this specific epitope instead of the epitope of a CD3-binding molecule previously described in bispecific T cell-engaging molecules, do not nonspecifically activate T cells to the same extent as observed with previous generation T cell-engaging antibodies. This reduced T cell activation is associated with less or reduced T cell redistribution in patients, which has been determined to pose a risk of side effects.

[0006] Currently, antibodies, including bispecific antibodies, are typically produced using a fed-batch manufacturing process. Fed-batch culture is a well-known biotechnology process technique in which one or more nutrients (culture medium) are fed into a bioreactor during cultivation, with the product remaining in the bioreactor until the end of the run (Tsuneo Yamane, Shoichi Shimizu: Fed-batch Techniques in Microbial Processes. (1984) Advances in Biochem Eng. / Biotechnol, 30:147-194). Therefore, bispecific antibody products are prone to accumulation during the fed-batch process, resulting in loss of product quality due to, for example, aggregation, clipping, or certain chemical degradation reactions. In addition, the product may not be obtained until the end of the run. In addition, process-related impurities, such as host cell proteins (HCPs), also accumulate in the bioreactor during the fed-batch process. Removal of these impurities downstream is often difficult, requiring additional measures and resources to ensure the quality of the final product. Because each new run requires a new cell culture growth phase, the required repeated growth phases impair the overall productivity of fed-batch. Furthermore, to achieve the sufficient production volumes produced by fed-batch facilities, large bioreactors are required, which use a large amount of space and energy. Therefore, improved upstream manufacturing processes specifically for bispecific antibody production are needed to improve both product quantity and product quality in order to provide sufficient amounts of product at commercial scale with a quality that reduces the amount of product that needs to be discarded in downstream processing. Given the cost of large-scale cell culture processes and the increasing demand for larger volumes and lower costs of biological products to be delivered to patients with severe unmet medical needs, new process methods that can incrementally improve recombinant protein production and recovery are valuable. In this regard, perfusion or continuous perfusion production of biopharmaceuticals in bioreactors is a promising strategy to improve process productivity, flexibility, and efficiency.Due to the operational complexity of continuous processes compared to perfusion or fed-batch processes, a higher degree of automation is required to obtain reliable results. To increase the robustness of higher biomass processes, an automated biomass-based feeding strategy is proposed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 99 / 54440 [Patent Document 2] International Publication No. 2005 / 040220 [Patent Document 3] International Publication No. 2008 / 119567 [Non-patent literature]

[0008] [Non-Patent Document 1] Fan,Gaowei;Wang,Zujian;Hao,Mingju;Li,Jinming(2015). “Bispecific antibodies and their applications”.Journal of Hematology & Oncology.8:130 [Non-patent document 2] Yang, Fa; Wen, Weihong; Qin, Weijun (2016). “Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies”. International Journal of Molecular Sciences. 18(1):48 [Non-patent document 3] Tsuneo Yamane, Shoichi Shimizu:Fed-batch Techniques in Microbial Processes.(1984)Advances in Biochem Eng. / Biotechnol,30:147-194 Summary of the Invention [Means for solving the problem]

[0009] Surprisingly, an adapted perfusion or continuous perfusion manufacturing process can be provided, including automated biomass-based controlled perfusion flow rates, ensuring a more efficient process that is automated and less prone to individual errors, for example, by operating staff. The process according to the present invention is therefore easier to manufacture and operate. In this regard, improved (bispecific) antibody product quality can be obtained. Even though continuous manufacturing processes for the production of proteins such as antibodies are known (e.g., Cattaneo et al., U.S. Patent Application Publication No. 2017 / 0204446 A1), such processes were not tailored to the specific needs of bispecific antibodies, which are prone to aggregation, clipping, and chemical degradation during upstream manufacturing process steps, thus resulting in reduced product quantity and quality. Adjusting the reactor biomass (cell concentration) is one of the primary means of realizing these gains. Proof-of-concept experiments have shown that while high biomass (up to 40%) with a constant feed rate results in lower viability (>75%), high biomass (up to 50%) with a biomass-based feed rate results in higher viability (>90%). It has also been found in the context of the present invention that by reaching and maintaining a higher biomass in the bioreactor, i.e., at least 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% packed cell volume (PCV; i.e., the percentage of solids in the cell suspension in the bioreactor), there is less lactate production, which is generally known to be detrimental to biotechnological production processes, as cells normally stop growing in a high lactate environment, meaning a loss of biomass and ultimately yield and productivity.

[0010] Thus, in one aspect, in the context of the present invention there is provided an upstream manufacturing process for the production of an antibody product that applies automated (as opposed to manual, i.e., non-automated) measurement and regulation of perfusion flow in a perfusion bioreactor (see FIG. 1 for overall apparatus), comprising: (i) providing a liquid cell culture medium comprising at least one mammalian cell culture in a perfusion bioreactor, wherein the mammalian cell culture is capable of expressing an antibody product and the cells have a concentration (viable cell density, VCD) of at least 1 x 10 cells / mL at the time of inoculation in the perfusion bioreactor; (ii) providing a first control loop for measuring and regulating medium level in the bioreactor, the first control loop including a level probe that measures the medium level in the bioreactor relative to a set point, a permeate pump calibrated to measure permeate volume (volume per time), and level control means that receives input from the level probe and the permeate pump and is capable of correcting the medium feed rate to the bioreactor in response to the medium pump (feed pump) in response to input from the level probe and the permeate probe, or the level control means that receives input from the level probe and the medium pump and is capable of correcting the output rate from the bioreactor in response to input from the level probe and the medium probe, wherein measurements of the medium level in the bioreactor are taken at predetermined fixed time intervals; (iii) providing a second control loop for measuring and regulating biomass in the bioreactor, the second control loop including a dielectric or Raman probe in the bioreactor that measures biomass, preferably the dielectric probe, and biomass control means that receives input from the biomass dielectric or Raman probe and is capable of correcting discharge rate from the bioreactor in response to a discharge pump in response to the input, wherein measurements of biomass in the bioreactor are taken at predetermined fixed time intervals; (iv) providing an integrated first and second control loop by connecting the biomass control means and the level control means to an integrated unit, the integrated unit being capable of performing an automated perfusion rate calculation, the perfusion rate being a function of the biomass value, preferably according to the formula: Perfusion rate (mL / min) = function of biomass values ​​(dielectric constant, PCV, VCD, spectroscopic measurements) and / or Perfusion rate [mL / min] = Perfusion rate based on dielectric constant [cm / pF / d] × Dielectric constant value [pF / cm] where the constant is the perfusion rate [1 / d] divided by the dielectric constant [pF / cm], and the dielectric constant value is 0.5-120 pF / cm during a first period (growth phase) during which the biomass in the bioreactor increases to approximately a predetermined biomass set point and / or 25-100 pF / cm during a second period (production phase) of biomass stabilization after the predetermined biomass set point is reached; and (v) automatically correcting or maintaining the perfusion rate by the integrated unit, which sends signals to the permeate pump and / or medium pump to increase or decrease the pumping rate, respectively, depending on the biomass measured at predetermined fixed time intervals. It is envisaged to provide a process comprising:

[0011] Within this aspect of the invention, in step (i), it is envisaged that the cells have a concentration of at least 7 x 10^5 cells / mL at the time of inoculation in the bioreactor.

[0012] Within this aspect of the invention, in step (iv), it is envisaged that the biomass set point is equal to a VCD of at least 30 x 10^6 cells / mL, preferably 30 x 10^6 cells / mL, if the manufacturing process is a fed-batch process, and equal to a VCD of 65 x 10^6 cells / mL if the manufacturing process is a continuous manufacturing process.

[0013] Within this aspect of the invention, it is envisaged that in step (iv), proliferation of the cell culture occurs for at least 4 days, preferably at least 7 days, more preferably at least 12 or 14 days.

[0014] Within this aspect of the invention, in step (ii), it is envisaged that the preset fixed time interval corresponds to a maximum of 1 minute, preferably 30 seconds, more preferably a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 seconds, preferably 1 second.

[0015] Within this aspect of the invention, in step (iii), it is envisaged that the preset fixed time interval corresponds to a maximum of 1 minute, preferably 30 seconds, more preferably a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 seconds, preferably 1 second.

[0016] Within this aspect of the invention, in step (v), it is envisaged that the preset fixed time interval corresponds to a maximum of 1 minute, preferably 30 seconds, more preferably a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 seconds, preferably 1 second.

[0017] Within this aspect of the invention, the dielectric constant during the growth phase is envisaged to be 0.70 to 120 pF / cm, preferably 0.73 to 70.7 pF / cm, more preferably 1 to 20 pF / cm or 100 to 117 pF / cm when the manufacturing process is a continuous manufacturing process.

[0018] Within this aspect of the invention, it is contemplated that the specific cell perfusion rate based on the dielectric constant is 0.01-0.049 cm / pF / d, preferably 0.015-0.04 cm / pF / d, more preferably 0.02-0.04 cm / pF / d, and most preferably 0.0266-0.04 cm / pF / d during the growth phase. For non-continuous production, the upper limit may be higher, e.g., up to 0.2 cm / pF / d, preferably up to 0.13 cm / pF / d.

[0019] Within this aspect of the invention, it is envisaged that the applied perfusion rate corresponds to a CSPR of 0.01-0.1 nL / cell / d during the proliferation phase, preferably 0.02-0.08 nL / cell / d, more preferably 0.027-0.076 nL / cell / d during the proliferation phase.

[0020] Within this aspect of the invention, it is envisaged that the dielectric constant in production will be between 55 and 85 pF / cm, preferably between 60 and 75 pF / cm, and more preferably between 62 and 73 pF / cm.

[0021] Within this aspect of the invention, the specific cell perfusion rate based on the dielectric constant is contemplated to be 0.01-0.04 cm / pF / d, preferably 0.01-0.035 cm / pF / d, and even more preferably 0.01-0.0266 cm / pF / d during the production phase.

[0022] Within this aspect of the invention, the applied perfusion rate is envisaged to correspond to a CSPR of 0.01-0.49 nL / cell / d, preferably 0.015-0.04 nL / cell / d, particularly preferably 0.023-0.035 nL / cell / d during the production phase.

[0023] Within this aspect of the invention, it is envisaged that the production period will be at least 14 days, preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 days, if the production process is a continuous manufacturing process, and at least 3 days, preferably 4 or 5 days, if the production process is a fed-batch process.

[0024] Within this aspect of the invention, it is envisioned that the upstream manufacturing process is a perfusion process (e.g., fed-batch) or a continuous perfusion (continuous manufacturing) process.

[0025] Within this aspect of the invention, it is envisaged that the antibody product is preferably a full-length antibody, such as a monoclonal antibody, directed against PD-1, e.g., an IgG antibody, or a non-full-length bispecific molecule.

[0026] Within this aspect of the invention, it is envisaged that the antibody product is a full length antibody or molecule, which is preferably based on a full length antibody or a fragment thereof, which is bispecific.

[0027] Within this aspect of the invention, it is contemplated that the antibody product is a fusion protein, preferably an anti-PD-1 mAb / IL-21 mutein fusion protein.

[0028] Within this aspect of the invention, it is envisaged that the antibody product is a bispecific non-full-length antibody molecule comprising a first binding domain and a second binding domain that bind to a target and an effector cell, respectively.

[0029] Within this aspect of the invention, it is envisaged that the bispecific molecule preferably comprises a half-life extending moiety selected from human serum albumin (HAS), an HAS binding domain or an Fc-based half-life extending moiety derived from an IgG antibody, most preferably an scFc half-life extending moiety.

[0030] Within this aspect of the invention, it is envisaged that the bispecific molecule is a bispecific T cell engager molecule.

[0031] Within this aspect of the invention, it is envisaged that the first binding domain of the bispecific molecule binds to at least one target cell surface antigen selected from the group consisting of CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, EpCAM, CD70, MUC17, CLDN18, BCMA and PSMA.

[0032] Within this aspect of the invention, it is envisaged that the second binding moiety of the bispecific antibody product binds to CD3.

[0033] Within this aspect of the invention, the first binding domain comprises: (a) CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5 and CDR-L3 as set forth in SEQ ID NO: 6; (b) CDR-H1 as set forth in SEQ ID NO: 29, CDR-H2 as set forth in SEQ ID NO: 30, CDR-H3 as set forth in SEQ ID NO: 31, CDR-L1 as set forth in SEQ ID NO: 34, CDR-L2 as set forth in SEQ ID NO: 35 and CDR-L3 as set forth in SEQ ID NO: 36; (c) CDR-H1 as set forth in SEQ ID NO: 42, CDR-H2 as set forth in SEQ ID NO: 43, CDR-H3 as set forth in SEQ ID NO: 44, CDR-L1 as set forth in SEQ ID NO: 45, CDR-L2 as set forth in SEQ ID NO: 46 and CDR-L3 as set forth in SEQ ID NO: 47; (d) CDR-H1 as set forth in SEQ ID NO: 53, CDR-H2 as set forth in SEQ ID NO: 54, CDR-H3 as set forth in SEQ ID NO: 55, CDR-L1 as set forth in SEQ ID NO: 56, CDR-L2 as set forth in SEQ ID NO: 57 and CDR-L3 as set forth in SEQ ID NO: 58; (e) CDR-H1 as set forth in SEQ ID NO: 65, CDR-H2 as set forth in SEQ ID NO: 66, CDR-H3 as set forth in SEQ ID NO: 67, CDR-L1 as set forth in SEQ ID NO: 68, CDR-L2 as set forth in SEQ ID NO: 69, and CDR-L3 as set forth in SEQ ID NO: 70; (f) CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, and CDR-L3 as set forth in SEQ ID NO: 88; (g) CDR-H1 as set forth in SEQ ID NO: 94, CDR-H2 as set forth in SEQ ID NO: 95, CDR-H3 as set forth in SEQ ID NO: 96, CDR-L1 as set forth in SEQ ID NO: 97, CDR-L2 as set forth in SEQ ID NO: 98 and CDR-L3 as set forth in SEQ ID NO: 99; (h) CDR-H1 as set forth in SEQ ID NO: 105, CDR-H2 as set forth in SEQ ID NO: 106, CDR-H3 as set forth in SEQ ID NO: 107, CDR-L1 as set forth in SEQ ID NO: 109, CDR-L2 as set forth in SEQ ID NO: 110, and CDR-L3 as set forth in SEQ ID NO: 111; (i) CDR-H1 as set forth in SEQ ID NO: 115, CDR-H2 as set forth in SEQ ID NO: 116, CDR-H3 as set forth in SEQ ID NO: 117, CDR-L1 as set forth in SEQ ID NO: 118, CDR-L2 as set forth in SEQ ID NO: 119, and CDR-L3 as set forth in SEQ ID NO: 120; (j) CDR-H1 as set forth in SEQ ID NO: 126, CDR-H2 as set forth in SEQ ID NO: 127, CDR-H3 as set forth in SEQ ID NO: 128, CDR-L1 as set forth in SEQ ID NO: 129, CDR-L2 as set forth in SEQ ID NO: 130, and CDR-L3 as set forth in SEQ ID NO: 131; (k) CDR-H1 as set forth in SEQ ID NO: 137, CDR-H2 as set forth in SEQ ID NO: 138, CDR-H3 as set forth in SEQ ID NO: 139, CDR-L1 as set forth in SEQ ID NO: 140, CDR-L2 as set forth in SEQ ID NO: 141 and CDR-L3 as set forth in SEQ ID NO: 142; (l) CDR-H1 as set forth in SEQ ID NO: 152, CDR-H2 as set forth in SEQ ID NO: 153, CDR-H3 as set forth in SEQ ID NO: 154, CDR-L1 as set forth in SEQ ID NO: 155, CDR-L2 as set forth in SEQ ID NO: 156, and CDR-L3 as set forth in SEQ ID NO: 157; (m) CDR-H1 as set forth in SEQ ID NO: 167, CDR-H2 as set forth in SEQ ID NO: 168, CDR-H3 as set forth in SEQ ID NO: 169, CDR-L1 as set forth in SEQ ID NO: 170, CDR-L2 as set forth in SEQ ID NO: 171 and CDR-L3 as set forth in SEQ ID NO: 172; (n) CDR-H1 as set forth in SEQ ID NO: 203, CDR-H2 as set forth in SEQ ID NO: 204, CDR-H3 as set forth in SEQ ID NO: 205, CDR-L1 as set forth in SEQ ID NO: 206, CDR-L2 as set forth in SEQ ID NO: 207 and CDR-L3 as set forth in SEQ ID NO: 208; (o) CDR-H1 as set forth in SEQ ID NO: 214, CDR-H2 as set forth in SEQ ID NO: 215, CDR-H3 as set forth in SEQ ID NO: 216, CDR-L1 as set forth in SEQ ID NO: 217, CDR-L2 as set forth in SEQ ID NO: 218, and CDR-L3 as set forth in SEQ ID NO: 219; (p) CDR-H1 as set forth in SEQ ID NO: 226, CDR-H2 as set forth in SEQ ID NO: 227, CDR-H3 as set forth in SEQ ID NO: 228, CDR-L1 as set forth in SEQ ID NO: 229, CDR-L2 as set forth in SEQ ID NO: 230, and CDR-L3 as set forth in SEQ ID NO: 231; and (q) CDR-H1 as set forth in SEQ ID NO: 238, CDR-H2 as set forth in SEQ ID NO: 239, CDR-H3 as set forth in SEQ ID NO: 240, CDR-L1 as set forth in SEQ ID NO: 241, CDR-L2 as set forth in SEQ ID NO: 242, and CDR-L3 as set forth in SEQ ID NO: 243. It is envisioned that the antibody comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, and a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of:

[0034] Within this aspect of the invention, it is envisioned that the perfusion culture is conducted continuously for at least 7 days, preferably at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, and most preferably at least 35 days, by supplying a defined cell specific perfusion rate and withdrawing excess cells from the bioreactor to maintain the biomass set point.

[0035] In a second aspect of the present invention, it is envisaged to provide an apparatus for carrying out the continuous upstream manufacturing process of claim 1, comprising a perfusion bioreactor, a first control loop, a second control loop and an integrated unit.

[0036] In a third aspect, the present invention envisages providing a (bispecific) antibody product produced by the upstream manufacturing process of claim 1. [Brief explanation of the drawings]

[0037] [Figure 1] 1 shows one arrangement of an automated continuous manufacturing process according to the present invention, where (i.) the permeate pump is controlled by a level control that receives input from a level probe and a feed pump, or (ii.) the feed pump is controlled by a level control that receives input from a level probe and a permeate pump. The second case (ii.) is preferably used in embodiments of the present invention. [Figure 2] Figure 1 shows the viability (A), PCV (B), permittivity specific perfusion rate (CSPR) (C), and biomass specific perfusion rate (BSPR, (D)) and cell culture values ​​during the production phase of the continuous perfusion process. The control process had a manual time-based feed rate [volume / day] and a fixed PCV. The test conditions had a manual PCV-based feed rate [volume / day / %PCV] and a manually increased PCV setpoint. The test conditions resulted in good cell growth; i.e., the PCV was increased to 50% (after day 23) with high viability. The desired setpoint was 0.078 l / day from days 23 to 33. [Figure 3]Metabolite values ​​(lactate (A), osmolality (B), glucose (C), and ammonia (D)) during the production phase of a continuous perfusion process with manual PCV-based feeding [volume / day / PCV] are shown. The control process had a manual time-based feeding [volume / day] and a fixed PCV. The test condition had a manual PCV-based feeding [volume / day / %PCV] and a manually increased PCV setpoint. Metabolite trends were similar between the test and control conditions. [Figure 4] Viability (A), PCV (B), CSPR (C), and BSPR (D) using automated dielectric constant-based perfusion rate [cm / pF.day] control during the growth phase of a continuous perfusion process are shown. The control process had manual time-based feeding [volume / day] during the growth phase. The test conditions had automated dielectric constant-based feeding (0.04 cm / pF.day on days 4-10 and 0.03 cm / pF.day on days 11-14). The test conditions resulted in good cell growth. [Figure 5] Metabolite values ​​(lactate (A), osmolality (B), glucose (C), and ammonia (D)) utilizing automated permittivity-based perfusion rate [cm / pF.day] control during the growth phase of the continuous perfusion process are shown. The control process had a manual time-based feed rate [volume / day] during the growth phase. The test condition had an automated permittivity-based feed (0.04 cm / pF.day on days 4-10 and 0.03 cm / pF.day on days 11-14). Metabolite trends were similar between the test and control conditions. [Figure 6] The CSPR (A) and BSPR (B), PCV (C), and permeate volume (D) during the production phase of the continuous perfusion process are shown. There was a fixed feed rate [volume / day] from days 12 to 26. There was automated permittivity-based perfusion flow control at a rate of 0.0277 cm / pF.day from days 27 to 32. There were three PCV setpoints tested throughout the experiment from days 12 to 32 (19%, 23%, and 27%). PCV was more tightly controlled with the automated permittivity-based feed from days 27 to 32 compared to the fixed feed rate from days 12 to 26. [Figure 7]Cell culture values ​​for product concentration (titer) (A) and cell viability (B), as well as lactate (C) and osmolality (D) in the permeate during the production phase of the continuous perfusion process, are shown. A fixed feed rate [volume / day] was used from days 12 to 26. Automated dielectric constant-based perfusion flow control was used at a rate of 0.0277 cm / pF / day from days 27 to 32. Three PCV setpoints were tested throughout the experiment from days 12 to 32 (19%, 23%, and 27%). Higher titers were observed at higher PCV setpoints in both the fixed feed rate and dielectric constant-based feed conditions. At a fixed BSPR from days 27 to 32, titer, lactate, and osmolality were more stable. Meanwhile, at a fixed feed rate from days 12 to 26, titer increased over time. [Figure 8] Figure 1 shows the VCD (A), production volume (B), viability (C), and product concentration (titer) in the permeate (D) during both the expansion and production phases of continuous manufacturing for a CD70xCD3 bispecific T-cell engaging molecule. The control condition (triangle symbols) had a fixed feed rate [volume / day]. Test conditions had automated dielectric constant-based perfusion rates during the expansion and production phases at three levels: high (cross symbols; 0.065 cm / pF / day on days 0–6, 0.035 cm / pF / day on days 7–12), medium (dash symbols; 0.03, 0.017), and low (circle symbols; 0.02, 0.01). The maximum rate resulted in higher production, titer, and lower viability in non-steady-state cell culture conditions. The minimum rate resulted in steady-state cell culture operation with stable viability, but lower production and titer. The medium speed and control had similar performance to each other, which was between the performance of the high and low speeds. [Figure 9]Figure 1 shows productivity (A), product concentration (titer) (B), VCD (C), and viability (D) in the perfusion process for the PD1xIL21 mutein antibody construct. Control and background conditions had a fixed feed rate [volume / day]. Test conditions had automated dielectric constant-based perfusion rates during the growth and production phases (0.12 cm / pF / day on days 3-8 and 0.03 cm / pF / day on days 9-15). Test conditions had higher VCD and viability, lower and / or similar productivity to control and background conditions. [Figure 10-1] Figure 1 shows productivity (A), product concentration (titer) (B), VCD (C), and viability (D) in a perfusion process for PD1 mAb. Control and background conditions had a fixed feed rate [volume / day]. Test conditions had automated dielectric constant-based perfusion rates during the growth and production phases (0.12 cm / pF / day on days 3-8 and 0.03 cm / pF / day on days 9-15). Test conditions had higher productivity, lower titer, VCD, and viability compared to background conditions. [Figure 10-2] Figure 1 shows productivity (A), product concentration (titer) (B), VCD (C), and viability (D) in a perfusion process for PD1 mAb. Control and background conditions had a fixed feed rate [volume / day]. Test conditions had automated dielectric constant-based perfusion rates during the growth and production phases (0.12 cm / pF / day on days 3-8 and 0.03 cm / pF / day on days 9-15). Test conditions had higher productivity, lower titer, VCD, and viability compared to background conditions. DETAILED DESCRIPTION OF THE INVENTION

[0038] Provided herein is a perfusion or continuous perfusion process for producing biologics, i.e., therapeutic proteins, particularly antibodies and bispecific molecules. The present invention contemplates tailoring an upstream process to the specific requirements of bispecific antibody production. The upstream process not only contributes to increased productivity and reduced space requirements compared to standard fed-batch manufacturing solutions known in the art, but also anticipates that the continuous manufacturing process (preferably a continuous upstream manufacturing process) is specifically adapted for bispecific antibodies and results in higher product quality, i.e., a higher monomer content relative to fed-batch manufacturing, and reduced aggregated bispecific antibodies. In particular, the method according to the present invention does not require new components or new equipment, but instead utilizes a novel combination of known components and equipment, and new, very high-frequency online measurements and process controls that respond directly to those measurements, to facilitate automation of the continuous manufacturing process, typically in a time of up to one minute, preferably as little as one second.

[0039] The present invention is based on the precise and timely measurement of biomass during the growth and production phases to control the production process of biopharmaceuticals such as antibodies, antibody constructs, or bispecific T-cell engager molecules. Biomass is preferably measured in the context of the present invention by biodielectric permittivity measurements, but may also be measured manually offline. However, Raman spectroscopy is also envisaged in the context of the present invention as an advantageous method for determining biomass by Raman probe and, where appropriate, controlling the production process of the biopharmaceutical of interest. Cell cultures controlled according to the present invention can typically be controlled as steady-state in continuous perfusion or in non-steady-state mode in continuous or discontinuous perfusion processes.

[0040] In the context of this invention, Raman spectroscopy is understood as a spectroscopic technique that utilizes Raman scattering, or inelastic scattering, of monochromatic laser light to study rotational, vibrational, and other modes of a system. The interaction between phonons and the laser light results in an energy shift, which provides information about the state of the phonons in the system. Typically, laser light is used to illuminate a sample. Electromagnetic radiation from the laser spot is collected by a lens and passed through a collimator. Molecules are excited from their ground state to an excited state and then relax to a vibrationally excited state. This results in Stokes Raman scattering. If the molecule is already in a vibrational state, it is called anti-Stokes Raman scattering. A change in polarizability is required for the molecule to exhibit Raman scattering. While the intensity of Raman scattering depends on the change in polarizability, the Raman shift is based on the associated vibrational levels. Advanced versions of Raman spectroscopy include stimulated Raman spectroscopy, surface-enhanced Raman spectroscopy, and resonance Raman spectroscopy. Raman spectroscopy results can be related to biomass and provide biomass measurements.

[0041] In the context of the present invention, dielectric constant (usually denoted by the Greek letter ε (epsilon)) is a measure of the electronic polarizability of a dielectric material. Materials such as the (outer) membrane of a (living) cell with a high dielectric constant polarize more in response to an applied electric field than materials with a low dielectric constant, thereby storing more energy in the field. Therefore, dielectric constant plays an important role in determining capacitance as described herein. Typically, the electric displacement D resulting from an applied electric field E is D=εE. More generally, dielectric constant is a thermodynamic function of state. It may depend on the frequency, amplitude, and direction of the applied field. The SI unit for dielectric constant is farads per meter (F / m). The capacitance of a capacitor is based on its design and construction, which means that it does not change with charging and discharging. The formula for capacitance in a parallel plate capacitor is:

number

[0042] The present process does not require the extra step of converting biopermittivity values ​​(pF / cm) to viable cell density (VCD - 1E6 cells / mL) values ​​as in prior art methods. Similarly, the constant applied is technically not cell specific perfusion rate (CSPR - mL / 1E6 cells.day), but rather permittivity specific perfusion rate (cm / pF.day), again in contrast to typical methods found in the prior art.

[0043] In the context of the present invention, a cell coefficient of 1 is used. It is not altered for the cell line, which in the context of the present invention is usually a CHO cell. As proposed in the prior art, it may be possible to correlate the biodielectric constant with the VCD, but this model will also depend on the cell diameter, which may vary. The biodielectric constant will correlate better with the biomass, as measured by packed cell volume (PCV). Here, the PCV measurement is used only to adjust the biodielectric constant set point for the continuous manufacturing process for the bispecific molecule instead of the cell coefficient.

[0044] In the prior art, Dowd et al. make hourly adjustments based on two biopermittivity measurements (converted to VCD using a model). The method according to the system of the present invention takes biopermittivity readings more frequently, such as every second, and calculates the perfusion (permeate) volume per second to obtain an optimized biomass specific perfusion rate (BSPR), which increases productivity, for example, by allowing a higher PCV and improving product quality by, for example, avoiding high lactate levels.

[0045] The constant in the formula applied by the integration unit is based on the dielectric constant (which would be CSPR, not cell density). The unit of the constant is cm / pF / day. This is derived by dividing the permeate volume (1 / day) by the dielectric constant (pF / cm). The constant or constants according to the present invention will work for any given molecule. The constant may depend on the cell line, medium, and molecule stability.

[0046] The method according to the invention has been found to have several advantages over methods described in the prior art. The process is simpler, without the need for an extra VCD model. The applied constants can be changed for each molecule / process / cell line / media. However, there is no need to calculate the cell coefficient for each new process. Furthermore, the process according to the invention does not introduce errors in estimating the VCD. If the cell diameter increases due to some adverse process event, the VCD model will become inaccurate. Also, the process according to the invention is more sensitive to changes in biomass, since the second control loop typically measures every second. Advantageously, no instabilities were observed due to the simpler integration of the control loop.

[0047] In the context of the present invention, it is particularly advantageous to provide balanced process parameters that are particularly suitable for biologics such as the bispecific molecules as described herein. Typically, a lower CSPR results in higher productivity, but the CSPR must not fall below a lower limit as described herein, as once the minimum value is reached, viability drops sharply, making the process unable to continue and resulting in failure. For example, for the CD70xCD3 bispecific molecules as described herein, 0.01 nL / cell / d is preferred.

[0048] In the context of the present invention, an advantage of CSPR-based feeding is that biomass (and therefore productivity) can be preferably increased without significant impact on viability, as illustrated in Figure 2. Furthermore, in the context of the present invention, CSPR-based feeding had metabolite profiles at least similar to the control (manual), even though biomass was typically increased, as illustrated in Figure 3.

[0049] A certain low product concentration in the bioreactor crucially contributes to the avoidance of aggregates, i.e., a higher relative and / or absolute monomer concentration in the product. This is essential to ensure product quality and improve the overall economics of the process. The fewer aggregates generated upstream, the less rejects need to be removed downstream. Product concentrations below 3.5 g / L are associated with a low probability of aggregation. Product quality is further improved if the maximum product concentration throughout the upstream process is kept below 1.2 g / L. Product concentrations below 0.5 or even 0.3 g / L are even more preferred. By ensuring a sufficiently high perfusion rate of 1 vvd or preferably at least 2 vvd or more, economically advantageous production rates of preferably aggregate-free product can be achieved. This applies to all bispecific antibody products, whether they are full-length antibodies or non-full-length antibodies such as (single-chain) bispecific molecules.

[0050] Another surprising aspect in the context of the present invention is the fact that for bispecific antibody products, it is preferable to adapt the perfusion rate to the VCD in order to obtain a preferred product quality and quantity. In this regard, the perfusion rate is continuously, gradually, or incrementally increased after inoculation until a preferred setpoint is reached. Typically, this setpoint is reached when the biomass setpoint is equal to a mean viable cell density (VCD) of at least 35×10^6 cells / mL, preferably at least 65×10^6 cells / mL, more preferably at least 71×10^6 cells / mL, and most preferably at least 85×10^6 cells / mL. Typically, the perfusion rate is set to a low value as long as the VCD is low relative to the maximum VCD reached in the same process. For example, if the VCD is equal to approximately 0.5×10^6 cells, the perfusion rate may be as low as approximately 0.4 VCD. However, as VCD increases due to cell growth in the bioreactor, the perfusion rate can be increased gradually or incrementally, e.g., continuously, from 0.4 vvd to 2 vvd, until a biomass set point of, e.g., 35 x 10^6 cells / mL is reached. Preferably, the higher the perfusion rate, the higher the biomass set point. For example, when the biomass set point is, e.g., at least 65 x 10^6 cells / mL, more preferably at least 71 x 10^6 cells / mL, and most preferably at least 85 x 10^6 cells / mL, vvd can be set to at least 2, preferably at least 2.01, 3, 4, 5, 6, or even 6.4.

[0051] It is also envisaged in the context of the present invention that the perfusion rate is adjusted throughout the continuous manufacturing process in response to the continuously measured VCD. VCD is understood to be a readily available and reliable parameter. The integrated viable cell density (IVCD) is understood herein as the area under the curve for VCD as a function of time. Thereby, for example, it may be preferable to maintain a constant cell specific perfusion rate (CSPR, nL per cell per day), which may contribute to a controlled product concentration in the bioreactor to avoid negative effects on product quality.

[0052] As a result, controlled and preferably low product concentrations (e.g., preferably less than 1.2 g / l for full length bispecific antibodies, preferably less than 0.4 g / l for HLE bispecific molecules, and preferably less than 0.12 g / l for non-HLE bispecific molecules according to the invention) are ensured throughout the continuous upstream manufacturing process, resulting in less product affected by aggregation, clipping or other chemical degradation.

[0053] In the context of the present invention, CSPR is the average VCD (C V , i.e., the average number of viable cells per mL) to the perfusion volume D (bioreactor volume per day).

number

[0054] In addition, in the context of the present invention, it is understood that it is preferable to provide cells in cell culture with a consistent microenvironment regardless of cell density. Therefore, it is preferable to exchange the medium at a rate proportional to the cell density. By applying a perfusion rate based on a preferred CSPR, a perfusion rate suitable for the cell density can be obtained.

[0055] In the context of the present invention, CSPR may preferably be applied automatically by a control station with online biomass measurements, for example based on biodielectric constants or Raman spectroscopy. VThis allows for minute and / or stable regulation of D in response to fluctuations in D. Such a stable, i.e., continuous, response may be preferred instead of stepwise, i.e., incremental or discontinuous, changes in D. The minimum CSPR is the amount that delivers the minimum amount of nutrients to meet cellular needs and supports high productivity. The application of a minimum or near-minimum CSPR is of particular practical importance in high cell densities, e.g., high-cell-density cultivation (HCDC). In the context of the present invention, HCDC targets cell cultures having a VCD of, for example, at least 65 x 10^6 cells / mL, preferably at least 71 x 10^6 cells / mL, or even at least 85 x 10^6 cells / mL. It is also envisioned that HCDC may have a VCD of at least 100 x 10^6 cells / mL.

[0056] A typical minimum CSPR in the context of the present invention is 0.01 nl / cell / day. Within the preferred boundaries common to all bispecific molecules or antibodies as contemplated herein, some have even more preferred values ​​for best product quality and / or quantity. For example, in the context of the present invention, the CSPR of the CD19xCD3 BiTE® molecule is preferably less than 0.04 nl / cell-day, more preferably 0.028 nl / cell / day or less. For bispecific molecules containing an I2C domain targeting CD3 (SEQ ID NO: 26), such as the CD33xCD3 BiTE® molecule, the CSPR is preferably 0.028 nl / cell-day or less, or at least 0.051 nl / cell-day, more preferably 0.06-0.1 nl / cell / day. For full-length bispecific antibodies, such as TNF-α × TL1A bispecific antibodies or PD1-blocking mAbs, the CSPR (CD-based CSPR) is preferably 0.028 nL / cell-day or less, preferably less than 0.2 nL / cell-day or at least 0.051 nL / cell-day, and more preferably 0.06-0.1 nL / cell-day. For bispecific molecules as disclosed herein, such as CD70 × CD3 bispecific T cell engager molecules, a lower dielectric constant-based CSPR value advantageously results in higher productivity compared to classical fed-batch production, at the expense of lower titers (see, e.g., Figure 9). Thus, the methods presented herein have been demonstrated to be suitable for automating and streamlining the production process of biologics, particularly bispecific T cell engager molecules, to conserve resources. At the same time, productivity can be increased.

[0057] In the context of the present invention, "cell culture" or "culturing" refers to the growth and propagation of cells outside of a multicellular organism or tissue. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid culture medium.

[0058] The term "mammalian cell" refers to any cell from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, the mammalian cell may be an immortalized cell. In some embodiments, the mammalian cell is a differentiated cell. In some embodiments, the mammalian cell is an undifferentiated cell. Non-limiting examples of mammalian cells are described herein. A preferred type of mammalian cell in the context of the present invention is a GS-KO cell. Further examples of mammalian cells are known in the art.

[0059] As used herein, the term "cell culture medium" (also referred to as "culture medium," "cell culture media," or "tissue culture medium") refers to any nutrient solution used for growing cells, e.g., animal or mammalian cells, and generally provides at least one or more of the following: an energy source (usually in the form of a carbohydrate such as glucose); all essential amino acids, and generally one or more of the 20 basic amino acids and cysteine; vitamins and / or other organic compounds, which are typically required in low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements, which are typically required in very low concentrations, usually in the micromolar range.

[0060] Cell culture media include, but are not limited to, those commonly utilized in and / or known for use with any cell culture process, such as batch, expanded batch, fed-batch, and / or perfusion or continuous culture of cells.

[0061] "Growth" cell culture medium or feed medium refers to a cell culture medium used in cell culture during the period of exponential growth, the "growth phase," and is sufficiently complete to support cell culture during this phase. Growth cell culture medium may also contain a selection agent that confers resistance or survival to a selectable marker incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, cell culture medium lacking glycine, hypoxanthine and thymidine, or cell culture medium lacking only thymidine.

[0062] "Production" cell culture medium or feed medium refers to a cell culture medium typically used in cell cultures during the transition phase when exponential growth ends and during the subsequent transition and / or production phase when protein production becomes dominant. Such cell culture medium is sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase.

[0063] "Perfusion" cell culture medium or feed medium refers to a cell culture medium typically used in cell culture that is maintained by perfusion or continuous culture methods and is complete enough to support cell culture during this process. Perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate the method used to remove spent medium. Perfusion cell culture medium may be used in both the growth and production phases.

[0064] The term "0.5x amount" means about 50% of the amount. The term "0.6x amount" means about 60% of the amount. Similarly, 0.7x, 0.8x, 0.9x, and 1.0x mean about 70%, 80%, 90%, or 100% of the amount, respectively.

[0065] The term "culture" or "cell culture" refers to the maintenance or growth of mammalian cells under a controlled set of physical conditions.

[0066] The term "mammalian cell culture" means a liquid culture medium containing a plurality of mammalian cells maintained or grown under a controlled set of physical conditions.

[0067] The term "liquid culture medium" refers to a liquid containing sufficient nutrients to grow or proliferate cells (e.g., mammalian cells) in vitro. For example, the liquid culture medium may contain one or more of amino acids (e.g., the 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvic acid, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium may contain mammalian serum. In some embodiments, the liquid culture medium does not contain mammalian serum or another extract (defined liquid culture medium). In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Another example of a liquid culture medium is a minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture media are described herein. Further examples of liquid culture media are known in the art and commercially available. The liquid culture medium can contain mammalian cells at any density. For example, as used herein, the volume of liquid culture medium removed from the bioreactor can be substantially free of mammalian cells.

[0068] In the context of the present invention, a "bioreactor" refers to a vessel suitable for carrying out perfusion cell culture in which at least steps (i) to (iii) of the present invention are carried out. The bioreactor may be a disposable vessel made of, for example, a plastic material, or a reusable vessel made of, for example, stainless steel.

[0069] The term "agitation" means stirring or otherwise moving a portion of the liquid culture medium in a bioreactor. This is done, for example, to increase the dissolved O concentration in the liquid culture medium in the bioreactor. Agitation can be done using any method known in the art, for example, using an instrument or propeller. Exemplary devices and methods that can be used to achieve agitation of a portion of the liquid culture medium in a bioreactor are known in the art.

[0070] The term "continuous process" refers to a process in which a liquid is continuously fed through at least a portion of a system. For example, in any of the exemplary continuous biological manufacturing systems described herein, a liquid culture medium containing a recombinant therapeutic protein is continuously fed into the system, and a therapeutic protein drug substance is fed from the system while the system is running.

[0071] The term "fed-batch bioreactor" is a term of art and refers to a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein culturing the cells present in the bioreactor involves periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without substantial or significant removal of either the first or second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is added as a dry powder.

[0072] The term "clipping" refers to the partial cleavage of an expressed protein, usually by proteolysis.

[0073] The term "degradation" generally refers to the breakdown of a larger entity, such as a peptide or protein, into at least two smaller entities, one of which may be significantly larger than the other.

[0074] The term "deamidation" refers to any chemical reaction in which an amide functionality, typically in the side chain of an amino acid such as asparagine or glutamine, is removed or converted to another functionality. Usually, asparagine is converted to aspartic acid or isoaspartic acid.

[0075] The term "aggregation" generally refers to direct mutual attraction between molecules, for example, via van der Waals forces or chemical bonds. In particular, aggregation is understood as proteins accumulating and clumping together. Aggregates may include amorphous aggregates, oligomers, and amyloid fibrils, and are typically referred to as high molecular weight (HMW) species, i.e., molecules having a higher molecular weight than the pure product molecules, which are typically non-aggregated molecules, also referred to herein as low molecular weight (LMW) species or monomers.

[0076] Acidic species are generally understood herein to be included among the variants commonly observed when analyzing antibodies by charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX). These variants are referred to as acidic or basic species relative to the predominant species. When antibodies are analyzed using IEF-based methods, acidic species are typically variants with a low apparent pI, and basic species are variants with a high apparent pI.

[0077] The dielectric constant probe according to the present invention is preferably an Incyte probe, which applies an AC electric field to the culture and measures the resulting polarization and depolarization of live cells and microorganisms by dielectric constant readings (permittivity per area). This signal can be related to viable cell density, since only viable cells can be polarized. Dead cells have leaky membranes and cannot be polarized. Therefore, the method is insensitive to dead cells, cell debris, and microcarriers.

[0078] Incyte is a dielectric-based sensor that responds only to living cells.

[0079] The term "residence time" generally refers to the time a particular product molecule resides in a bioreactor, i.e., the time ranging from its biotechnological production to its separation from the lumen of the bioreactor.

[0080] "Product quality" is typically assessed by the presence or absence of clipping, degradation, deamidation, and / or aggregation. For example, a product (molecule) containing a percentile content of HMW species of less than 40%, preferably less than 35%, or even less than 30%, 25%, or 20% may be considered to be of favorable product quality. Also, favorable product quality is accompanied by an essential absence of residual host cell proteins (HCPs) and clipping, degradation, and deamidation, or a significant reduction in HCP concentration, clipping, degradation, and / or deamidation, compared to products produced by processes other than those of the present invention, such as fed-batch processes. Methods known in the art for assessing product quality in the context of the present invention include cation exchange high-performance chromatography (CEX-HPLC) for charge variant analysis, tryptic peptide mapping for chemical modifications, host cell protein (HCP) ELISA, reduced capillary electrophoresis-sodium dodecyl sulfate (RCE-SDS), and size-exclusion high-performance liquid chromatography (SE-HPLC).

[0081] The term "antibody product" refers to a "secreted protein" or "secreted recombinant protein," meaning a protein (e.g., a recombinant protein) that originally contains at least one secretory signal sequence when translated in a mammalian cell and that is secreted, at least in part, into the extracellular space (e.g., liquid culture medium) via enzymatic cleavage of the secretory signal sequence within the mammalian cell. One of skill in the art will understand that a "secreted" protein need not completely dissociate from the cell to be considered a secreted protein.

[0082] The term bispecific antibody product encompasses bispecific antibodies such as full-length, e.g., IgG-based antibodies and fragments thereof, which are generally referred to herein as bispecific antibody molecules.

[0083] The term "antibody construct", or alternatively bispecific T cell engager molecule or bispecific molecule, refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length or complete immunoglobulin molecule (usually composed of two non-truncated heavy chains and two light chains), and / or derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. An antibody construct is therefore capable of binding to its specific target or antigen. Furthermore, a domain that binds to a binding partner according to the present invention is herein understood as a binding domain of an antibody construct according to the present invention. Typically, a binding domain according to the present invention comprises the minimum structural requirements of an antibody that enable target binding. This minimum requirement may be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. An alternative way of defining the minimal structural requirements of an antibody is by defining the epitope of the antibody within the structure of a specific target, protein domains of the target protein (epitope clusters) that each constitute an epitope region, or by reference to specific antibodies that compete with the epitope of the defined antibody. Antibodies on which the constructs according to the invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies.

[0084] The binding domain of an antibody construct or bispecific T cell engager molecule according to the invention may, for example, comprise the CDRs of the above-referenced groups. Preferably, the CDRs are comprised within the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), although it does not have to comprise both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of an intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants, or binding domains include: (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment, which has two VH and CH1 domains; (4) an Fv fragment, which has the VL and VH domains of one arm of an antibody; (5) a dAb fragment, which has a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fvs (scFvs), the latter of which is preferred (e.g., derived from an scFv library).

[0042] Exemplary embodiments of antibody constructs or bispecific molecules according to the invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910 and WO 2015 / 048272.

[0085] The definition of "binding domain" or "domain that binds to" also includes fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "rIgG" ("half antibodies"). The antibody construct or bispecific T cell engager molecule according to the invention may also include engineered 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, comprising only one variable domain which may be VHH, VH or VL, which specifically binds to an antigen or epitope independently of other V regions or domains.

[0086] As used herein, the term "single-chain Fv," "single-chain antibody," or "scFv" refers to a single polypeptide chain antibody fragment that contains the variable regions from both the heavy and light chains, but lacks the constant region. Typically, single-chain antibodies further contain a polypeptide linker between the VH and VL domains that enables them to form the desired structure that enables antigen binding. 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 making single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 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; 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.

[0087] Furthermore, the definition of the term "antibody construct" or bispecific T cell engager molecule includes monovalent, bivalent and polyvalent / multivalent constructs, and therefore bispecific constructs that specifically bind to only two antigenic structures, as well as polyspecific / multispecific constructs that specifically bind to more than two antigenic structures, for example three, four or more, through different binding domains. Furthermore, the definition of the term "antibody construct" or bispecific T cell engager molecule includes molecules that consist of only one polypeptide chain, as well as molecules that consist of two or more polypeptide chains, where the chains can be either identical (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heterooligomers). Examples of the above-identified antibodies and variants or derivatives thereof are described, inter alia, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Duebel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0088] As used herein, the term "bispecific" refers to an antibody construct that is "at least bispecific," i.e., it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (here, a surface antigen on a target cell) and the second binding domain binds to another antigen or target (e.g., CD3). Thus, the antibody construct according to the present invention has specificity for at least two different antigens or targets. Furthermore, the bispecific T cell engager molecules according to the present invention are specifically characterized by targeting two different cells, i.e., effector T cells and target cells, at one time and combining them to achieve a therapeutic effect. Thus, the term "bispecific" in the context of the present invention particularly refers to dual cell targeting, i.e., targeting and combining two different cells. For example, the first domain preferably does not bind to one or more extracellular epitopes of CD3ε of the species described herein. The term "surface antigen on a target cell" refers to an antigenic structure expressed by a cell and present on its cell surface in a manner accessible to the antibody constructs described herein. It 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. Preferably, it is a tumor antigen. The term "bispecific antibody construct" according to the present invention also encompasses multispecific antibody constructs, such as trispecific antibody constructs comprising three binding domains or constructs with four or more (e.g., four, five, ...) specificities.

[0089] When antibody constructs or bispecific molecules according to the present invention are (at least) bispecific, they do not occur in nature and differ significantly from naturally occurring products. Thus, a "bispecific" antibody construct or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sites with different specificities. Bispecific antibody constructs or bispecific molecules can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0090] The at least two binding domains and variable domains (VH / VL) of the antibody construct or bispecific 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 interconnects the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antibody construct or bispecific molecule of the present invention. A peptide linker can also be used to fuse a third domain to another domain of the antibody construct of the present invention. The essential technical feature of such a peptide linker is that it does not contain any polymerization activity. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344. A peptide linker can also be used to connect other domains, modules, or regions (such as half-life extending domains) to the antibody construct of the present invention.

[0091] The antibody construct of the present invention is preferably an "in vitro generated antibody construct." This term refers to an antibody construct as defined above in which all or a part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection, such as in vitro phage display, protein chip, or any other method that allows testing of candidate sequences for antigen-binding ability. Thus, this term preferably excludes sequences generated solely by genome rearrangement in immune cells of an animal. A "recombinant antibody" is an antibody generated by using recombinant DNA technology or genetic engineering.

[0092] As used herein, the term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, and are therefore uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0093] For the preparation of monoclonal antibodies, any technique that results in antibodies produced by continuous cell line cultures can be used. For example, the monoclonal antibodies used can be made by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Additional 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).

[0094] The hybridomas can then 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 the designated antigen. Any form of the relevant antigen can be used as the immunogen, including, for example, recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof. Surface plasmon resonance, as employed in the BIAcore system, can be used to increase the efficiency of phage antibody binding to epitopes on surface antigens of target cells (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0095] Another exemplary method for generating monoclonal antibodies involves screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in U.S. Patent No. 5,223,409 to Ladner et al.; 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).

[0096] In addition to using display libraries, the relevant antigen can be used to immunize a non-human animal (e.g., a rodent (e.g., a mouse, hamster, rabbit, or rat)). In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, mouse strains deficient in mouse antibody production can be engineered with large fragments of the human Ig (immunoglobulin) locus. Hybridoma technology can be used to generate and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0097] Monoclonal antibodies can also be obtained from non-human animals and then modified, e.g., humanized, deimmunized, chimerized, etc., using recombinant DNA techniques known in the art. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with modified effector function (see, e.g., U.S. Pat. No. 5,648,260; Kontermann and Duebel (2010), supra; and Little (2009), supra).

[0098] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for an antigen during an immune response. Repeated exposure to the same antigen leads the host to produce antibodies with successively increasing affinities. Similar to natural prototyping, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within CDRs can be introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. 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.

[0099] A preferred type of amino acid substitution variant of an antibody construct involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties relative 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 displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domain and, for example, a surface antigen on a human target cell. Such contact and adjacent residues are candidates for substitution using the techniques detailed herein. After generating such variants, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0100] The monoclonal antibodies and antibody constructs of the present invention include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkey, ape, etc.) and human constant region sequences. Various methods for producing chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. Sci USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0101] Antibodies, antibody constructs, antibody fragments, or antibody variants can also be modified by specific deletion of human T-cell epitopes (a method called "deimmunization") by methods disclosed in the Examples of WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II. These peptides represent potential T-cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). To detect potential T-cell epitopes, a computer modeling method called "peptide threading" can be applied, as described in WO 98 / 52976 and WO 00 / 34317. In addition, databases of human MHC class II-binding peptides can be searched for motifs present in VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and therefore represent potential T cell epitopes. Potential T cell epitopes detected can be eliminated by substituting a small number of amino acid residues within the variable domains, or preferably by single amino acid substitutions. Conservative substitutions are usually made. In many, but not all, amino acids common to positions within human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. 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 VBASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (edited by Tomlinson, L.A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs.For example, the consensus human framework regions described in US Pat. No. 6,300,064 can be used.

[0102] "Humanized" antibodies, antibody constructs, variants, or fragments thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins of largely human sequence that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also called CDR) of the recipient are replaced by residues from a hypervariable region of a non-human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a 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).

[0103] Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; and U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of immunoglobulin Fv variable domains 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 predetermined target, as described above. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0104] Humanized antibodies can also be produced using transgenic animals, such as mice, that express human heavy and light chain genes but are incapable of expressing endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that can be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR, or only some of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

[0105] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such modified immunoglobulin molecules can be produced 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 EP 239400).

[0106] The terms "human antibody," "human antibody construct," and "human binding domain" include antibodies, antibody constructs, and binding domains having antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) supra. Human antibodies, antibody constructs, or binding domains of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. A human antibody, antibody construct, or binding domain may have at least one, two, three, four, five, or more positions substituted with an amino acid residue not encoded by human germline immunoglobulin sequences. However, as used herein, the definitions of human antibody, antibody construct, and binding domain also contemplate "fully human antibodies" which comprise only human sequences of non-artificially and / or genetically modified antibodies, which may be obtained by using technologies or systems such as Xenomouse. Preferably, a "fully human antibody" does not contain amino acid residues that are not encoded by human germline immunoglobulins.

[0107] In some embodiments, the antibody construct of the present invention is an "isolated" or "substantially pure" antibody construct. "Isolated" or "substantially pure," when used to describe an antibody construct disclosed herein, refers to an antibody construct that has been identified, separated, and / or recovered from components of its production environment. Preferably, the antibody construct is free or substantially free from association with all other components from its production environment. Contaminating components of its production environment, such as components arising from recombinant transfected cells, are materials that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An antibody construct may, for example, constitute 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 an isolated protein may constitute 5% to 99.9% by weight of the total protein content, depending on the circumstances. The use of inducible promoters or high-expression promoters to produce the polypeptide at high concentration levels can allow for production of significantly higher concentrations of the polypeptide. This definition includes production of antibody constructs in a variety of organisms and / or host cells known in the art. In preferred embodiments, the antibody construct will be purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Typically, however, an isolated antibody construct will be prepared by at least one purification step.

[0108] The term "binding domain" in the context of the present invention is considered to be a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target site on a target molecule (antigen), such as CD33 and CD3, respectively. The structure and function of the first binding domain (e.g., recognizing CD33), and preferably also the structure and / or function of the second binding domain (e.g., recognizing CD3), are based on the structure and / or function of an antibody, e.g., a full-length or complete immunoglobulin molecule, and / or are derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or a fragment thereof. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). The second binding domain preferably also comprises the minimal structural requirements of an antibody that enable target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VH region). It is envisaged that the first and / or second binding domains may be generated or obtained by phage display or library screening methods other than grafting CDR sequences from an existing (monoclonal) antibody onto a scaffold.

[0109] According to the present invention, a binding domain is in the form of one or more polypeptides. Such polypeptides may comprise proteinaceous and non-proteinaceous portions (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise two or more amino acids linked together through covalent peptide bonds (resulting in a chain of amino acids).

[0110] As used herein, the term "polypeptide" generally refers to a group of molecules consisting of more than 30 amino acids. Polypeptides may further form multimers, such as dimers, trimers, and higher oligomers, i.e., consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its native form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally modified peptides / polypeptides / proteins, e.g., modified by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, "peptide," "polypeptide," or "protein" may also be chemically modified, such as by pegylation. Such modifications are well known in the art and are described herein below.

[0111] Preferably, the binding domain that binds to the surface antigen of a target cell and / or the binding domain that binds to CD3ε are human binding domains. Antibodies and antibody constructs comprising at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs with non-human variable and / or constant regions, such as those derived from rodents (e.g., mouse, rat, hamster, or rabbit). The presence of such rodent-derived proteins can lead to rapid clearance of the antibody or antibody construct or can generate an immune response against the antibody or antibody construct by the patient. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody function into rodents such that the rodents produce fully human antibodies.

[0112] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents will provide unique insights into the expression and regulation of nascent human gene products, their transfer to other systems, and their involvement in disease induction and progression.

[0113] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B cell development. Furthermore, such a strategy would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), a key milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antibody constructs are expected to minimize the immunogenicity and allergic reactions inherent in mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of administered antibodies / antibody constructs. The use of fully human antibodies or antibody constructs is expected to offer significant advantages in the treatment of chronic and recurrent human diseases that require repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0114] One approach toward this goal has been to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci, with the expectation that such mice would produce a broad repertoire of human antibodies without producing mouse antibodies. Large human Ig fragments would preserve the broad diversity of variable genes and the appropriate regulation of antibody production and expression. By utilizing the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoire recapitulated in these mouse strains should produce high-affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity could be readily generated and selected. This general strategy was demonstrated in connection with the generation of the first XenoMouse mouse strains (see Green et al., Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered with yeast artificial chromosomes (YACs) containing 245-kb and 190-kb germline-configured fragments of the human heavy chain and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. These human Ig-containing YACs proved compatible with the mouse system for both antibody rearrangement and expression, and were able to replace 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 to generate antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing multiple V genes, additional regulatory elements, and human Ig constant regions, could recapitulate a virtually complete repertoire characteristic of the human humoral response to infection and immunization. Recently, extending the work of Green et al., the introduction of megabase-sized germline-configured YAC fragments of the human heavy chain and kappa light chain loci introduced approximately 80% of the human antibody repertoire.See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application Publication No. 08 / 759,620.

[0115] The generation of XenoMouse mice is described in U.S. patent application Ser. Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837, 08 / 48 and 6,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752, and 08 / 759,620; and U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181, and 5,939,598, and Japanese Patent Publications Nos. 3068180B2, 3068506B2, and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0116] In another approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In this minilocus approach, an exogenous Ig locus is mimicked by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) form a construct that is inserted into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al., and U.S. Pat. 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; and 5,877,397 to Lonberg and Kay, respectively. Nos. 5,874,299; and 6,255,458 to Krimpenfort and Berns, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharm and U.S. Patent Application Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International.See also EP 0546073B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, and U.S. Pat. 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), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0117] Kirin has also demonstrated the production of human antibodies from mice into which large chromosome fragments or entire chromosomes have been introduced by microcell fusion. See European Patent Applications Nos. 773288 and 843961. Xenerex Biosciences is developing a technology for the potential production of human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes, e.g., B cells and / or T cells. The mice are then immunized with an antigen and can generate an immune response against that antigen. See U.S. Patent Nos. 5,476,996; 5,698,767; and 5,958,765.

[0118] Human anti-mouse antibody (HAMA) responses have driven the industry to create chimeric or otherwise humanized antibodies. However, it is expected that some human anti-chimeric antibody (HACA) responses will be observed, particularly with chronic or multi-dose antibody use. Therefore, it would be desirable to provide antibody constructs that contain a human binding domain for a target cell surface antigen and a human binding domain for CD3ε to eliminate the concerns and / or impact of HAMA or HACA responses.

[0119] The terms "(specifically) bind", "(specifically) recognize", "(specifically) be attracted to" and "(specifically) react" mean, according to the present invention, that a binding domain interacts or specifically interacts with a target molecule (antigen), here a surface antigen of a target cell and a given epitope or a given target site on CD3ε, respectively.

[0120] The term "epitope" refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and therefore the term epitope is also sometimes referred to herein as an "antigenic structure" or "antigenic determinant." The binding domain is therefore an "antigen interaction site." It is understood that said binding / interaction also defines "specific recognition."

[0121] An "epitope" can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. A "linear epitope" is an epitope that comprises an epitope recognized by a primary amino acid sequence. Linear epitopes typically contain at least three or at least four, and more usually at least five, or at least six, or at least seven, e.g., about 8 to about 10, amino acids in a unique sequence.

[0122] In contrast to linear epitopes, a "conformational epitope" is an epitope in which the primary sequence of amino acids comprising the epitope is not the only element defining the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by a binding domain). Generally, a conformational epitope comprises a larger number of amino acids than a linear epitope. In recognizing a conformational epitope, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is contained within the surface antigen protein of a target cell). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones forming the conformational epitope are juxtaposed, thereby enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.

[0123] The epitope mapping method is described below. When a region (a contiguous stretch of amino acids) of a human target cell surface antigen protein is exchanged / substituted with a corresponding region of a non-human or non-primate target cell surface antigen (e.g., a mouse target cell surface antigen, but also chicken, rat, hamster, rabbit, etc.), a reduction in the binding activity of the binding domain is expected, as long as the binding domain is not cross-reactive with the non-human or non-primate target cell surface antigen used. This 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 the binding to the corresponding region in the human target cell surface antigen protein, assuming binding to the corresponding region in the human target cell surface antigen protein to be 100%. It is envisioned that the above-mentioned human target cell surface antigen / non-human target cell surface antigen chimeras are expressed in CHO cells. It is also envisaged that the human target cell surface antigen / non-human target cell surface antigen chimera is fused to the transmembrane and / or cytoplasmic domain of a different membrane-associated protein, such as EpCAM.

[0124] In an alternative or additional method of epitope mapping, several truncations of the extracellular domain of a human target cell surface antigen can be generated to determine the specific region recognized by the binding domain. In these truncations, different extracellular target cell surface antigen domains / subdomains or regions are deleted stepwise, starting from the N-terminus. It is contemplated that the truncated target cell surface antigen can be expressed in CHO cells. It is also contemplated that the truncated target cell surface antigen may be fused to the transmembrane and / or cytoplasmic domain of a different membrane-bound protein, such as EpCAM. It is also contemplated that the truncated target cell surface antigen may include a signal peptide domain at their N-terminus, such as a signal peptide derived from the mouse IgG heavy chain signal peptide. It is further contemplated that the truncated target cell surface antigen may include a v5 domain at the N-terminus (following the signal peptide), which can confirm their correct expression on the cell surface. It is expected that reduced or lost binding will occur for truncated target cell surface antigens that no longer encompass the target cell surface antigen region recognized by the binding domain. The reduction in binding is preferably at least 10%, 20%, 30%, 40% or 50%, more preferably at least 60%, 70%, 80% and most preferably 90%, 95% or even 100%, relative to binding to the entire surface antigen protein (or its extracellular region or domain) of a human target cell, taken as 100.

[0125] Another method for determining the contribution of specific residues of target cell surface antigens to recognition by antibody constructs or binding domains is alanine scanning, in which each analyzed residue is substituted with alanine, for example, by site-directed mutagenesis (see, e.g., Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7). Alanine is used because it mimics the secondary structure criteria of many other amino acids, yet is not bulky and has a chemically inert methyl functional group. If it is desirable to preserve the size of the mutated residue, bulky amino acids such as valine or leucine may sometimes be used. Alanine scanning is a mature technique that has been used for a long time.

[0126] The interaction between a binding domain and an epitope or an epitope-containing region means that the binding domain exhibits measurable affinity for the epitope / epitope-containing region on a particular protein or antigen (herein, target cell surface antigen and CD3, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than target cell surface antigen or CD3. "Measurable affinity" means 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 -9Binding is considered specific when M is M. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reactivity of the binding domain to a target protein or antigen with the reactivity of the binding domain to proteins or antigens other than the target cell surface antigen or CD3. Preferably, the binding domains of the present invention essentially or substantially do not bind to proteins or antigens other than the target cell surface antigen or CD3 (i.e., the first binding domain cannot bind to proteins other than the target cell surface antigen, and the second binding domain cannot bind to proteins other than CD3). Superior affinity characteristics compared to other HLE formats are anticipated as a feature of the antibody constructs of the present invention. Such superior affinity consequently suggests an extended in vivo half-life. The longer half-life of the antibody constructs of the present invention may reduce the duration and frequency of administration, which usually contributes to improved patient compliance. This is particularly important because the antibody constructs of the present invention are particularly beneficial for highly debilitated or even multi-disease cancer patients.

[0127] The terms "does not essentially / substantially bind" or "cannot bind" mean that the binding domain of the invention does not bind to proteins or antigens other than target cell surface antigens or CD3, i.e., when binding to target cell surface antigens or CD3, respectively, is taken as 100%, it does not show 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 to proteins or antigens other than target cell surface antigens or CD3.

[0128] Specific binding is believed to be mediated by specific motifs within the amino acid sequences of the binding domain and the antigen. Thus, binding occurs as a result of their primary, secondary, and / or tertiary structures, as well as secondary modifications of said structures. The specific interaction of the antigen-interaction site with its specific antigen can result in simple binding of said site to the antigen. Furthermore, the specific interaction of the antigen-interaction site with its specific antigen can alternatively or additionally result in the initiation of a signal, for example, by inducing a conformational change in the antigen, oligomerization of the antigen, etc.

[0129] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is responsible for determining the specificity and binding affinity of a particular antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen-binding site.

[0130] The variability is not distributed uniformly throughout the variable domains of antibodies, but is concentrated in subdomains of each 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 domains are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs in three-dimensional space that form the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4) that largely adopt a β-sheet configuration, connected by three hypervariable regions that form loops connecting and, in some cases, forming part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs and contribute to the formation of the antigen-binding site with the hypervariable regions of the other chain (see Kabat et al., supra).

[0131] The term "CDR" and its plural "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 of which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). The CDRs contain most of the residues responsible for specific interactions between the antibody and the antigen and thus contribute to the functional activity of the antibody molecule. The CDRs are the primary determinants of antigen specificity.

[0132] The precise definition of CDR boundaries and lengths follows various classification and numbering systems. Thus, CDRs may be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering systems described herein. Although the boundaries differ, each of these systems has some overlap in the portions that constitute the so-called "hypervariable regions" within the variable sequences. Thus, CDR definitions according to these systems may differ in length and in the boundaries relative to the adjacent framework regions. See, e.g., Kabat (an approach based on sequence variability between species), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol., 1987, 196:901-917; and MacCallum et al., J. Mol. 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). As long as two residue identification techniques define overlapping but not identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

[0133] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main-chain conformation adopted by the antigen-binding (CDR) loop. Comparative structural studies have found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability found in the majority of the loops (see Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol., 1996, 263:800). Furthermore, there is a relationship between the loop structure adopted and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop and within the conserved framework (i.e., outside the loop), and therefore assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0134] The term "canonical structure" can also include considerations of the linear sequence of an antibody, for example, as classified by Kabat (Kabat et al., supra). The Kabat numbering scheme is a widely adopted standard for numbering amino acid residues in antibody variable domains in a consistent manner and is the preferred scheme applied in the present invention, as noted elsewhere herein. Additional structural considerations may also be used to determine the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering system can be described by the Chothia et al. numbering system 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 that allows, among other things, the identification of an appropriate chassis sequence (e.g., based on the desire to include various canonical structures in a library). The Kabat numbering system for antibody amino acid sequences and the structural considerations described in Chothia et al. (supra) and their significance for interpreting canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a general overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0135] The CDR3 of the light chain and especially the CDR3 of the heavy chain may be the most important determinant of antigen binding within the light and heavy chain variable regions. In some antibody constructs, the heavy chain CDR3 is likely to be the main contact area between the antigen and the antibody. Using an in vitro selection scheme that changes only the CDR3, the binding properties of the antibody can be changed or which residues contribute to antigen binding can be determined. Therefore, the 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.

[0136] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the 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") domains are not directly involved in antigen binding but exhibit various effector functions, such as antibody-dependent and cell-mediated cytotoxicity and complement activation. The Fc region of an antibody is contained within the heavy chain constant domain and can interact, for example, with Fc receptors located on the cell surface.

[0137] The sequences of antibody genes after construction and somatic mutation are highly diverse, and these diverse genes are 10 It is predicted that these genes encode 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 derived in whole or in part from at least one sequence encoding at least one immunoglobulin. The sequence may be generated by in vivo rearrangement of the V, D, and J segments of heavy chains and the V and J segments of light chains. Alternatively, the sequence may be generated from cells in response to, for example, in vitro stimuli that cause rearrangement. Alternatively, some or all of the sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, e.g., U.S. Pat. No. 5,565,332). A repertoire may include only one sequence or may include multiple sequences, including those within a genetically diverse collection.

[0138] The term "Fc portion" or "Fc monomer" in the context of the present invention refers to a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having the function of a CH3 domain of an immunoglobulin molecule. As is evident from the term "Fc monomer," a polypeptide comprising these CH domains is a "polypeptide monomer." An Fc monomer may be a polypeptide comprising a fragment of an immunoglobulin constant region excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but retaining a functional portion of at least one CH2 domain and one CH3 domain, with the CH2 domain located amino-terminal to the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region, and a CH3 region, with the hinge region located amino-terminal to the CH2 domain. The hinge region of the present invention is expected to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by papain digestion of an immunoglobulin region (which, of course, produces a dimer of two Fc polypeptides). In another aspect of this definition, an Fc monomer can be a polypeptide region comprising a portion of a CH2 region and a CH3 region. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by pepsin digestion of an immunoglobulin molecule. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequence of an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgM Fc region, an IgA Fc region, an IgD Fc region, and an IgE Fc region. (See, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because some variation exists 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 flexible hinge N-terminal to these domains.In the case of IgA and IgM, the Fc monomer may comprise a J chain. In the case of IgG, the Fc portion comprises immunoglobulin domains CH2 and CH3, and the hinge between the first two domains and CH2. Although the boundaries of the Fc portion may vary, an example of a human IgG heavy chain Fc portion comprising functional hinge, CH2, and CH3 domains can be defined, for example, according to Kabat, to include residues D231 (in the hinge domain—corresponding to D234 in Table 1 below) at the carboxyl terminus of the CH3 domain to P476, L476 (for IgG4), respectively. Two Fc portions or Fc monomers fused together via a peptide linker define the third domain of the antibody construct of the invention, which may also be defined as an scFc domain.

[0139] In one embodiment of the present invention, it is envisaged that the scFc domains disclosed herein, the Fc monomers each fused to one another, are comprised only in the third domain of the antibody construct.

[0140] In accordance with the present invention, IgG hinge regions can be identified by similarity using the Kabat numbering set forth in Table 1. In line with the above, it is envisaged that the hinge domain / region of the present invention comprises amino acid residues corresponding to the stretch of IgG1 sequence from D234 to P243 according to the Kabat numbering. Similarly, it is envisaged that the hinge domain / region of the present invention comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 182) (corresponding to the D234 to P243 section as set out in Table 1 below - variants of said sequence are also envisaged, provided that the hinge region still promotes dimerization). In a preferred embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain within the third domain of the antibody construct is eliminated by an N314X substitution, where X is any amino acid other than Q. Said substitution is preferably an N314G substitution. In a more preferred embodiment, said 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).

[0141] It is also envisioned that the third domain of the antibody construct of the invention comprises or consists of, in amino to carboxyl order: DKTHTCPPCP (SEQ ID NO: 182) (i.e., hinge)-CH2-CH3-linker-DKTHTCPPCP (SEQ ID NO: 182) (i.e., hinge)-CH2-CH3. The peptide linker of the aforementioned antibody construct is, in a preferred embodiment, 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 equal to or greater than 5 (e.g., 5, 6, 7, 8, etc., or more), with 6 being preferred ((Gly4Ser)6). The construct may further comprise the substitution N314X, preferably N314G, as described above, and / or the additional substitutions V321C and R309C. In a preferred embodiment of the antibody construct of the invention as defined herein above, it is envisaged that the second domain binds to an extracellular epitope of the human and / or Macaca CD3 epsilon chain.

[0142] [Table 1]

[0143] In further embodiments of the invention, the hinge domain / region comprises or consists of 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 have the following sequence EPKSCDKTHTCPPCP (as shown in Table 1 and in SEQ ID NO: 183). Accordingly, these core hinge regions are also envisaged in the context of the present invention.

[0144] The locations and sequences of the IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering set forth in Table 2.

[0145] [Table 2]

[0146] In one embodiment of the present invention, the amino acid residues highlighted in bold within the CH3 domain of the first or both Fc monomers are deleted.

[0147] The peptide linker by which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomer") are fused to one another preferably comprises at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferred that the linker comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, and most preferably exactly 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 equal to or greater than 5 (e.g., 6, 7, or 8). Preferably, the integer is 6 or 7, more preferably, the integer is 6.

[0148] 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 the first and second domains retain their distinct binding specificities independently of each other. For peptide linkers connecting at least two binding domains (or two variable domains) in the antibody construct of the present invention, these linkers preferably contain only a few amino acid residues, e.g., 12 or fewer amino acid residues. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Contemplated peptide linkers with fewer than 5 amino acids contain 4, 3, 2, or 1 amino acid, with Gly-rich linkers being preferred. A preferred embodiment of a peptide linker for fusing the first and second domains is shown in SEQ ID NO: 1. Preferred linker embodiments of peptide linkers for fusing the second and third domains are (Gly)4-linkers and G4-linkers, respectively.

[0149] A particularly preferred "single" amino acid in connection with one of the above "peptide linkers" is Gly. Thus, the above peptide linker may consist of a single amino acid, Gly. In a preferred embodiment 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 equal to or greater than 1 (e.g., 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1-12. Characteristics of the peptide linkers, including not promoting secondary structure, 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, peptide linkers that do not promote any secondary structure are preferred. The interconnection of the domains can be provided, for example, by genetic engineering as described in the Examples. Methods for preparing fused, operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0150] In a preferred embodiment of the antibody construct of the present invention, the first and second domains form an antibody construct of a format selected from the group consisting of (scFv)2, scFv-single domain mAb, diabody and oligomers of any of these formats.

[0151] According to a particularly preferred embodiment, and as described in the accompanying Examples, the first and second domains of the antibody construct of the present invention are "bispecific single-chain antibody constructs," more preferably bispecific "single-chain Fvs" (scFvs). Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker, as described herein above, which allows them to be produced, using recombinant methods, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, e.g., Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as intact or full-length antibodies. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable region of an immunoglobulin heavy chain (VH) and the variable region of an immunoglobulin light chain (VL), connected by a short linker peptide, typically about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of 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.

[0152] Bispecific single-chain antibody constructs are known in the art and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loeffler, Blood, (2000), 95, 6, 2098-2103, Bruehl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778; Kontermann and Duebel (2010), supra; and Little (2009), supra) can be adapted to produce single chain antibody constructs that specifically recognize a target of choice.

[0153] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv having the format (scFv)2) can be created by linking two scFv molecules (e.g., using a linker as described hereinabove). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably referred to as bivalent (i.e., it has two valencies for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably referred to as bispecific. Linking can be done by creating a single peptide chain with two VH and two VL regions to generate tandem scFvs (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to generate scFv molecules with a linker peptide that is too short (e.g., about 5 amino acids) to allow the two variable regions to fold together, resulting in dimerization of the scFv, a form known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0154] In accordance with the present invention, either the first domain, the second domain, or the first and second domains may comprise a single domain antibody, a variable domain of a single domain antibody, or at least the CDRs of a single domain antibody, respectively. Single domain antibodies contain only one (monomeric) antibody variable domain that is capable of selectively binding to a specific antigen independently of other V regions or domains. The first single domain antibodies were generated from heavy chain antibodies found in camels, and these consisted of V H The cartilaginous fish also have heavy chain antibodies (IgNAR), which are then called V fragments. NARSingle domain antibodies, called fragments, can be obtained. An alternative approach is to split the dimeric variable domain of a common immunoglobulin, for example, from a human or rodent, into monomers, thereby obtaining VH or VL as a single domain Ab. Most research on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single domain antibodies are called sdAbs, nanobodies, or single variable domain antibodies.

[0155] Therefore, (single domain mAb)2 is V H , V L , V H H and V NAR A monoclonal antibody construct is a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies independently selected from the group comprising: (a) an "scFv-single domain mAb" and (b) an "scFv-single domain mAb"; (b) an "scFv-single domain mAb"; (c) an "scFv-single domain mAb"; (d) an "scFv-single domain mAb"; (e) an "scFv-single domain mAb"; (f) an "scFv-single domain mAb"; (g) an "scFv-single domain mAb"; (h) an "scFv-single domain mAb"; (i) an "scFv-single domain mAb"; (j) an "scFv-single domain mAb"; (j) an "scFv-single domain mAb"; (k ...

[0156] The binding of an antibody construct to another given antibody construct in competition can be measured by a competitive assay, such as a competitive ELISA or a cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Similar to an avidin-coated ELISA plate, each of these beads can be used as a substrate when reacted with biotinylated proteins, on which the assay can be performed. The antigen is coated onto the beads, which are then pre-coated with the first antibody. A secondary antibody is added to confirm any further binding. Possible reading methods include flow cytometry.

[0157] T cells or T lymphocytes are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several subsets of T cells, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T cell receptors (TCRs) on their cell surface. The TCR is responsible for recognizing 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 an antigen peptide and MHC (peptide / MHC complex), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0158] The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains the CD3γ (gamma) chain, the 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 closely related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif, or ITAM, which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans. The most preferred CD3 epsilon epitope is contained within amino acid residues 1 to 27 of the human CD3 epsilon extracellular domain. The antibody constructs according to the invention are typically and advantageously expected to exhibit only minimal unspecific T cell activation, which is undesirable in specific immunotherapy, which translates into a low risk of side effects.

[0159] Lysis of redirected target cells via recruitment of T cells by multispecific, or at least bispecific, antibody constructs involves the formation of a cytolytic synapse and the delivery of perforin and granzymes. Engaged T cells are capable of continuous target cell lysis and are unaffected by immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, e.g., WO 2007 / 042261).

[0160] The cytotoxicity mediated by the antibody constructs of the present invention can be measured in various ways. Effector cells can 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 (at least the extracellular domain of) the surface antigen of the target cell, e.g., a surface antigen of a human or macaque target cell. The target cells can be a cell line (e.g., CHO) that has been stably or transiently transfected with the surface antigen of the target cell, e.g., a surface antigen of a human or macaque target cell. Alternatively, the target cells can be a cell line positively expressing the surface antigen of the natural target cell. Typically, EC 50 Values ​​are expected to be lower for target cell lines that express high levels of target cell surface antigen on the cell surface. The effector to target cell (E:T) ratio is usually about 10:1, but this can be varied. The cytotoxic activity of target cell surface antigen x CD3 bispecific antibody constructs is 51Cytotoxicity can be measured in a Cr release assay (approximately 18 hours of incubation) or a FACS-based cytotoxicity assay (approximately 48 hours of incubation). The incubation time (cytotoxic response) of the assay can be varied. Other methods for measuring cytotoxicity are well known to those skilled in the art and include MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques.

[0161] The cytotoxic activity mediated by the target cell surface antigen x CD3 bispecific antibody construct of the present invention is preferably measured in a cell-based cytotoxicity assay. 51 Cytotoxic activity can also be measured by EC 50 The EC50 value corresponds to the half-maximal effective concentration (the concentration of the antibody construct that induces a cytotoxic response halfway between the baseline and maximum). Preferably, the EC50 value corresponds to the half-maximal effective concentration (the concentration of the antibody construct that induces a cytotoxic response halfway between the baseline and maximum). 50 Values ​​are ≦5000 pM or ≦4000 pM, more preferably ≦3000 pM or ≦2000 pM, even more preferably ≦1000 pM or ≦500 pM, even more preferably ≦400 pM or ≦300 pM, even more preferably ≦200 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦20 pM or ≦10 pM, and most preferably ≦5 pM.

[0162] In various assays, the EC given above 50 Values ​​can be measured for stimulated / enriched CD8 + When T cells are used as effector cells, ECs are significantly higher than unstimulated PBMCs. 50 Those skilled in the art will recognize that values ​​can be expected to be lower. 50 Values ​​can be expected to be lower if the target cells express a large number of target cell surface antigens compared to rats with fewer target antigens. For example, stimulated / enriched human CD8 +When T cells are used as effector cells (and either target cell surface antigen-transfected cells such as CHO cells or target cell surface antigen-positive human cell lines are used as target cells), EC of the target cell surface antigen x CD3 bispecific antibody construct is used. 50 The value is preferably ≦1000 pM, more preferably ≦500 pM, even more preferably ≦250 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦10 pM, and most preferably ≦5 pM. When human PBMCs are used as effector cells, the EC of the target cell surface antigen x CD3 bispecific antibody construct is 50 The value is preferably ≦5000 pM or ≦4000 pM (especially when the target cells are a target cell surface antigen-positive human cell line), more preferably ≦2000 pM (especially when the target cells are cells transfected with the target cell surface antigen, such as CHO cells), more preferably ≦1000 pM or ≦500 pM, even more preferably ≦200 pM, even more preferably ≦150 pM, even more preferably ≦100 pM, and most preferably ≦50 pM or less. When a macaque T cell line, such as LnPx4119, is used as the effector cell and a cell line transfected with the surface antigen of a macaque target cell, such as CHO cells, is used as the target cell line, the EC of the target cell surface antigen x CD3 bispecific antibody construct is 50 The value is preferably ≦2000 pM or ≦1500 pM, more preferably ≦1000 pM or ≦500 pM, even more preferably ≦300 pM or ≦250 pM, even more preferably ≦100 pM, and most preferably ≦50 pM.

[0163] Preferably, the target cell surface antigen x CD3 bispecific antibody construct of the present invention does not induce / mediate lysis or essentially does not induce / mediate lysis of target cell surface antigen-negative cells, such as CHO cells. The terms "does not induce lysis," "does not essentially induce lysis," "does not mediate lysis," or "does not essentially mediate lysis" mean that, when the lysis of a target cell surface antigen-positive human cell line is taken as 100%, the antibody construct of the present invention does not induce or mediate lysis of more than 30%, preferably 20% or less, more preferably 10% or less, and particularly preferably 9%, 8%, 7%, 6%, or 5% or less of target cell surface antigen-negative cells. This is usually true at antibody construct concentrations up to 500 nM. Those skilled in the art will know how to measure cytolysis without further effort. Furthermore, specific instructions for measuring cytolysis are taught herein.

[0164] The difference in cytotoxic activity between the monomeric and dimeric isoforms of a particular target cell surface antigen x CD3 bispecific antibody construct is referred to as the "potency gap." This potency gap can be measured, for example, by comparing the EC 50 values ​​and EC of the dimeric form 50 The potency gap of the target cell surface antigen x CD3 bispecific antibody construct of the present invention is preferably ≦5, more preferably ≦4, even more preferably ≦3, even more preferably ≦2, and most preferably ≦1.

[0165] The first and / or second (or any further) binding domains of the antibody construct of the invention are preferably cross-species specific in members of the mammalian order Primates. Cross-species specific CD3 binding domains are described, for example, in WO 2008 / 119567. According to one embodiment, the first and / or second binding domains, in addition to binding to a surface antigen on a human target cell and human CD3, will also bind to a surface antigen / CD3 on a target cell of a primate, including, but not limited to, New World primates (such as marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus)), Old World primates (such as baboons and macaques), gibbons, and non-human homininae.

[0166] In one embodiment of the antibody construct of the invention, the first domain binds to a surface antigen of a human target cell and further binds to a surface antigen of a macaque target cell, such as a surface antigen of a cynomolgus monkey (Macaca fascicularis) target cell, more preferably a surface antigen of a macaque target cell expressed on the surface of a macaque cell. The affinity of the first binding domain for the surface antigen of a macaque target cell 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, and most preferably ≦0.05 nM or even ≦0.01 nM.

[0167] Preferably, the binding affinity gap of an antibody construct according to the present invention for the surface antigen of a macaque target cell to the surface antigen of a human target cell [surface antigen of a macaque target cell:surface antigen of a hu target cell] (determined, for example, by BiaCore or Scatchard analysis) is <100, preferably <20, more preferably <15, even more preferably <10, even more preferably <8, more preferably <6, and most preferably <2. A preferred range for the binding affinity gap of an antibody construct according to the present invention for the surface antigen of a macaque target cell to the surface antigen of a human target cell is 0.1-20, more preferably 0.2-10, even more preferably 0.3-6, even more preferably 0.5-3 or 0.5-2.5, and most preferably 0.5-2 or 0.6-2.

[0168] The second (binding) domain of the antibody construct of the invention binds to human CD3 epsilon and / or macaque CD3 epsilon. In a preferred embodiment, the second domain further binds to marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus) or squirrel monkey (Saimiri sciureus) CD3 epsilon. Both marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) are New World primates belonging to the marmoset (Callitrichidae) family, while squirrel monkeys (Saimiri sciureus) are New World primates belonging to the capuchin (Cebidae) family.

[0169] With respect to the antibody constructs of the present invention, the second binding domain that binds to an extracellular epitope of human and / or macaque CD3 comprises: (a) CDR-L1 as set forth in SEQ ID NO: 27 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 28 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 29 of WO 2008 / 119567; (b) CDR-L1 as set forth in SEQ ID NO: 117 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 118 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 119 of WO 2008 / 119567; and (c) Preferably, the VL region comprises CDR-L1, CDR-L2 and CDR-L3 selected from CDR-L1 as set forth in SEQ ID NO: 153 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 154 of WO 2008 / 119567 and CDR-L3 as set forth in SEQ ID NO: 155 of WO 2008 / 119567.

[0170] In a further preferred embodiment of the antibody construct of the invention, the second domain binding to an extracellular epitope of the human and / or macaque CD3 epsilon chain comprises: (a) CDR-H1 as set forth in SEQ ID NO: 12 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 13 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 14 of WO 2008 / 119567; (b) CDR-H1 as set forth in SEQ ID NO: 30 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 31 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 32 of WO 2008 / 119567; (c) CDR-H1 as set forth in SEQ ID NO: 48 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 49 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 50 of WO 2008 / 119567; (d) CDR-H1 as set forth in SEQ ID NO: 66 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 67 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 68 of WO 2008 / 119567; (e) CDR-H1 as set forth in SEQ ID NO: 84 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 85 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 86 of WO 2008 / 119567; (f) CDR-H1 as set forth in SEQ ID NO: 102 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 103 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 104 of WO 2008 / 119567; (g) CDR-H1 as set forth in SEQ ID NO: 120 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 121 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 122 of WO 2008 / 119567; (h) CDR-H1 as set forth in SEQ ID NO: 138 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 139 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 140 of WO 2008 / 119567; (i) CDR-H1 as set forth in SEQ ID NO: 156 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 157 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 158 of WO 2008 / 119567; and (j) a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from CDR-H1 as set forth in SEQ ID NO: 174 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 175 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 176 of WO 2008 / 119567.

[0171] In a preferred embodiment of the antibody construct of the invention, the above three sets of VL CDRs are combined with the above ten sets of VH CDRs in the second binding domain to form a set (30) comprising CDR-L1-3 and CDR-H1-3, respectively.

[0172] For the antibody constructs of the invention, it is preferred that the second domain that binds to CD3 comprises a VL region selected from the group consisting of the VL regions as set forth 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 WO2008 / 119567 or as set forth in SEQ ID NO: 200.

[0173] It is also preferred that the second domain that binds to CD3 comprises a VH region selected from the group consisting of the VH regions as set forth in SEQ ID NOs: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008 / 119567 or as set forth in SEQ ID NO: 201.

[0174] More preferably, the antibody construct of the invention comprises: (a) a VL region as set forth in SEQ ID NO: 17 or 21 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 15 or 19 of WO 2008 / 119567; (b) a VL region as set forth in SEQ ID NO: 35 or 39 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 33 or 37 of WO 2008 / 119567; (c) a VL region as set forth in SEQ ID NO: 53 or 57 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 51 or 55 of WO 2008 / 119567; (d) a VL region as set forth in SEQ ID NO: 71 or 75 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 69 or 73 of WO 2008 / 119567; (e) a VL region as set forth in SEQ ID NO: 89 or 93 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 87 or 91 of WO 2008 / 119567; (f) a VL region as set forth in SEQ ID NO: 107 or 111 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 105 or 109 of WO 2008 / 119567; (g) a VL region as set forth in SEQ ID NO: 125 or 129 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 123 or 127 of WO 2008 / 119567; (h) a VL region as set forth in SEQ ID NO: 143 or 147 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 141 or 145 of WO 2008 / 119567; (i) a VL region as set forth in SEQ ID NO: 161 or 165 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 159 or 163 of WO 2008 / 119567; and (j) a CD3-binding second domain comprising a VL region and a VH region selected from the group consisting of a VL region as set forth in SEQ ID NO: 179 or 183 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 177 or 181 of WO 2008 / 119567.

[0175] A CD3-binding second domain comprising a VL region as set forth in SEQ ID NO: 200 and a VH region as set forth in SEQ ID NO: 201 is also preferred in the context of an antibody construct of the invention.

[0176] According to a preferred embodiment of the antibody construct of the present invention, the first and / or second domain has the following format: a pair of VH and VL domains in the format of a single-chain antibody (scFv). The VH and VL domains are arranged in the order of VH-VL or VL-VH. It is preferred that the VH domain is arranged at the N-terminus of the linker sequence and the VL domain is arranged at the C-terminus of the linker sequence.

[0177] A preferred embodiment of the above-mentioned antibody construct of the present invention is characterized by a CD3-binding second domain 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 WO 2008 / 119567 or as set forth in SEQ ID NO: 202.

[0178] Covalent modifications of antibody constructs are also included within the scope of the present invention, which are generally, but not necessarily, carried out post-translationally. For example, some types of covalent modifications of antibody constructs are introduced into the molecule by reacting specific amino acid residues of the antibody construct with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0179] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0180] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0181] Arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with lysine and arginine epsilon-amino groups.

[0182] The specific modification of tyrosyl residues may be undertaken for the purpose of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane, most commonly using N-acetylimidizole and tetranitromethane to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 The chloramine T method described above, in which tyrosyl residues are iodinated with I to prepare labeled proteins for use in radioimmunoassay, is preferred.

[0183] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (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, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0184] Derivatization with bifunctional agents is useful for crosslinking the antibody constructs of the present invention to water-insoluble support matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include, for example, homobifunctional imidoesters including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as the ester with 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming crosslinks in the presence of light. Instead, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are used for protein immobilization.

[0185] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of this invention.

[0186] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0187] Another type of covalent modification of antibody constructs included within the scope of the invention involves altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below) and the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0188] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a sugar chain 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 the recognition sequences for enzymatic attachment of a sugar chain to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment 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.

[0189] Addition of glycosylation sites to an antibody construct is conveniently accomplished by altering the amino acid sequence to include one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). Briefly, it is preferred to alter the amino acid sequence of an antibody construct by alteration at the DNA level, in particular by mutating the DNA encoding the polypeptide at preselected bases to generate codons that will be translated into the desired amino acids.

[0190] Another means of increasing the number of carbohydrate moieties on an antibody construct is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell with glycosylation capabilities for N- and O-linked glycosylation. Depending on the linkage mode used, sugars can be added to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0191] Removal of carbohydrate moieties present on the starting antibody construct can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment cleaves most or all sugars except the linking 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 carbohydrate moieties on polypeptides can be achieved by the use of 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 described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin prevents the formation of protein-N-glycosidic bonds.

[0192] Other modifications of antibody constructs are also contemplated herein. For example, another type of covalent modification of an antibody construct includes linking the antibody construct to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, in the manner 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 antibody construct to facilitate the addition of polymers such as PEG.

[0193] In some embodiments, covalent modification of the antibody construct of the present invention includes the addition of one or more labels. To reduce potential steric hindrance, the labeling group may be attached to the antibody construct via a spacer arm of various lengths. Various methods for labeling proteins are known in the art and can be used in practicing the present invention. The term "label" or "labeling group" refers to any detectable label. Generally, labels are divided into various classes depending on the assay for detecting them, examples of which include, but are not limited to, those listed below. a) Radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 Isotopic labels, which can be radioactive isotopes or heavy isotopes, such as I) b) Magnetic labels (e.g., magnetic particles) c) redox-active moieties d) optical dyes (including but not limited to chromophores, fluorophores and fluorophores), such as fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), chemiluminescent groups and fluorophores, which can be either "small molecule" fluorophores or proteinaceous fluorophores; e) Enzymes (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.).

[0194] "Fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, 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 Suitable optical dyes, including fluorophores, include, but are not limited to, fluorescein ...

[0195] Suitable proteinaceous fluorescent labels include GFP from species of Renilla, Ptilosarcus, or Aequorea (Chalfie et al., 1994, Science 263:802-805), green fluorescent protein including EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), 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), enhanced 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 (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Pat. 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).

[0196] The antibody constructs of the present invention may also contain additional domains, e.g., useful for isolating the molecule or relevant for tailoring the pharmacokinetic profile of the molecule. Domains useful for isolating the antibody construct may be selected from peptide motifs or secondarily introduced moieties that can be captured by isolation methods, e.g., isolation columns. Non-limiting examples 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 variants thereof (e.g., Strep II tags), and His tags. All of the antibody constructs disclosed herein characterized by identified CDRs may contain a His tag domain, commonly known as a repeat of consecutive His residues, preferably five and more preferably six His residues (hexahistidine), in the amino acid sequence of the molecule. The His tag can be located, for example, at either the N- or C-terminus of the antibody construct, but is preferably located at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 199) is attached to the C-terminus of the antibody construct according to the present invention via a peptide bond. Additionally, PLGA-PEG-PLGA conjugate systems may be combined with polyhistidine tags for sustained release applications and improved pharmacokinetic profiles.

[0197] Amino acid sequence modifications of the antibody constructs described herein are also contemplated. For example, improving the binding affinity and / or other biological properties of the antibody construct may be desirable. Amino acid sequence variants of the antibody construct are produced by introducing appropriate nucleotide changes into the nucleic acid of the antibody construct or by peptide synthesis. All of the amino acid sequence modifications described below should result in an antibody construct that still retains the desired biological activity of the unmodified parent molecule (binding to target cell surface antigens and CD3).

[0198] The term "amino acid" or "amino acid residue" typically refers to an amino acid having an art-recognized definition, 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 (Ile 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), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified according to the presence of a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0199] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody construct. Any combination of deletion, insertion, and substitution may be made to arrive at the final construct, provided that the final construct retains the desired characteristics. Amino acid changes may also alter post-translational processes of the antibody construct, such as changing the number or location of glycosylation sites.

[0200] For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted, or deleted in each of the CDRs (depending, of course, on their length), 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 inserted, substituted, or deleted in each of the FRs. Preferably, insertions of amino acid sequences into the antibody construct include amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Corresponding modifications may be made within the third domain of the antibody construct of the invention. Insertional variants of the antibody construct of the invention include fusion of an enzyme or polypeptide to the N- or C-terminus of the antibody construct.

[0201] The most important sites for substitutional mutagenesis include, but are not limited to, the CDRs of the heavy and / or light chains, particularly the hypervariable regions, although modifications of the FRs in the heavy and / or light chains are also contemplated. 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 CDRs, 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 regions (FRs). For example, if the CDR sequence contains 6 amino acids, it is contemplated that 1, 2, or 3 of these amino acids may be substituted. Similarly, if the CDR sequence contains 15 amino acids, it is contemplated that 1, 2, 3, 4, 5, or 6 of these amino acids may be substituted.

[0202] A useful method for identifying specific residues or regions of an antibody construct that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells in Science, 244:1081-1085 (1989). In this method, a residue or target group of residues within the antibody construct (e.g., charged residues such as arg, asp, his, lys, and glu) that affect the interaction of the amino acid with the epitope is identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine).

[0203] Next, further or other variants are introduced at, or in place of, the substitution site to select those amino acid positions that demonstrate functional sensitivity to the substitution. Thus, the site or region for introducing an amino acid sequence variant is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze or optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be performed at the target codon or region, and the expressed antibody construct variants are screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites within DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using an assay for antigen binding activity, such as target cell surface antigen or CD3 binding.

[0204] Generally, when amino acids are substituted in one or more or all of the CDRs of the heavy and / or light chain, it is preferred that the resulting "substituted" sequence be 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 to the "substituted" sequence depends on the length of the CDR. For example, a CDR having five amino acids is preferably 80% identical to its substituted sequence, since it has at least one substituted amino acid. Thus, the CDRs of an antibody construct may have different degrees of identity to their substituted sequences, e.g., CDRL1 may have 80% while CDRL3 may have 90%.

[0205] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 3 below) is envisioned, as long as the antibody construct retains the ability to bind to a surface antigen on a target cell via the first domain and to CD3, CD3 epsilon via the second domain, respectively, and / or its CDRs have identity to the substituted sequences (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 sequences).

[0206] Conservative substitutions are shown under the heading of "preferred substitutions" in Table 3. If such substitutions alter biological activity, they are referred to as "exemplary substitutions" in Table 3, or more substantial changes as further described below in relation to amino acid classes can be introduced and the products screened for desired characteristics.

[0207] [Table 3]

[0208] Substantial alterations in the biological properties of the antibody constructs of the invention are achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone in the substituted region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk. 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.

[0209] Non-conservative substitutions would involve exchanging a member of one of these classes for another. Substitution of any cysteine ​​residue not involved in maintaining the proper conformation of the antibody construct, generally with serine, may improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, adding cysteine ​​bond(s) to an antibody may improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0210] For amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, including, 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 search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the BestFit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by visual inspection. Preferably, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0211] One example of a useful algorithm is PILEUP. PILEUP generates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a tree showing the clustering relationships used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method of 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 weighted end gaps.

[0212] 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. USA 90:5873-5787. 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 default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 1. The HSP S and HSP S2 parameters are dynamic values ​​established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched; however, the values ​​can be adjusted to increase sensitivity.

[0213] An additional useful algorithm is Gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score, the threshold T parameter is set to 9, the two-hit method resulting in an ungapped extension has a cost of 10+k for a gap length k, Xu is set to 16, and Xg is set to 40 for the database search stage and 67 for the output stage of the algorithm. Gapped alignments are initiated by a score corresponding to approximately 22 bits.

[0214] Generally, the amino acid homology, similarity, or identity between individual variant CDR or VH / VL sequences is at least 60% relative to the sequences set forth herein, and more typically, it is preferred that the homology or identity be increased 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 even closer to 100%. Similarly, "percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences of binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with nucleotide residues in the coding sequence of the antibody construct. A specific method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap coverage and overlap percentage of 1 and 0.125, respectively.

[0215] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding each variant CDR or VH / VL sequence and the nucleotide sequences set forth herein will be at least 60%, more typically 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 preferably increasing to nearly 100%. Thus, a "variant CDR" or "variant VH / VL region" is one that has particular homology, similarity or identity to a parent CDR / VH / VL of the invention and shares biological function, including but not limited to, at least 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.

[0216] In one embodiment, the percent identity of the antibody construct of the present invention to the human germline gene product is ≥70% or ≥75%, more preferably ≥80% or ≥85%, even more preferably ≥90%, and most preferably ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, or even ≥96%. Identity to the human antibody germline gene product is considered to be an important feature for reducing the risk of a therapeutic protein eliciting an immune response against the drug in patients undergoing treatment. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36 (2005) 3-10) demonstrate that reducing the non-human portion of a drug-antibody construct reduces the risk of eliciting anti-drug antibodies in patients undergoing treatment. Comparison of a large number of clinically evaluated antibody drugs and corresponding immunogenicity data has shown that humanization of antibody V regions tends to result in proteins that are less immunogenic (5.1% of patients on average) than antibodies with unmodified non-human V regions (23.59% on average). Therefore, for protein therapeutics in the form of V region-based antibody constructs, high identity to human sequences is desirable. To determine this germline identity, the V region of the 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 percentage of amino acid sequence can be calculated by dividing the number of identical amino acid residues by the total number of amino acid residues in the VL. A similar method can be used for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), with the exception that the VH CDR3 may be excluded due to its high diversity and lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase sequence identity to human antibody germline genes.

[0217] In a further embodiment, the bispecific antibody constructs of the invention exhibit high monomer yields under standard research scale conditions, for example in a standard two-step purification process. Preferably, the monomer yield of the antibody constructs according to the invention is ≥ 0.25 mg / L supernatant, more preferably ≥ 0.5 mg / L, even more preferably ≥ 1 mg / L, and most preferably ≥ 3 mg / L supernatant.

[0218] Similarly, the yield of dimeric antibody construct isoforms of the antibody construct and therefore the percentage of monomer (i.e., monomer:(monomer+dimer)) can be determined. Productivities of monomeric and dimeric antibody constructs and calculated percentage of monomer can be obtained, for example, by SEC purification of culture supernatants from standardized research-scale production in roller bottles. In one embodiment, the percentage of monomer of the antibody construct is ≧80%, more preferably ≧85%, even more preferably ≧90%, and most preferably ≧95%.

[0219] In one embodiment, the antibody construct preferably has a 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 antibody construct is determined by incubating the construct in human plasma at 37° C. for 24 hours, followed by 51The antibody can be tested by determining the EC50 in a chromium release cytotoxicity assay. The effector cells in the cytotoxicity assay can be stimulated enriched human CD8-positive T cells. The target cells can be, for example, CHO cells transfected with a surface antigen of a human target cell. The effector cell to target cell (E:T) ratio can be selected to be 10:1. The human plasma pool used for this purpose is derived from blood collected from healthy donors using an EDTA-coated syringe. Cellular components are removed by centrifugation, and the upper plasma phase is collected and then pooled. As a control, the antibody construct is 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).

[0220] It is further preferred that the antibody construct of the present invention has a low monomer-to-dimer conversion rate. The conversion rate can be measured under different conditions and analyzed by high-performance size exclusion chromatography. For example, incubation of the monomeric isoform of the antibody construct can be performed in an incubator at a concentration of, for example, 100 μg / ml or 250 μg / ml at 37° C. for 7 days. Under these conditions, the antibody construct of the present invention preferably exhibits a dimer fraction of ≦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%, ≦0.5%, or even 0%.

[0221] It is also preferred that the bispecific antibody constructs of the present invention exhibit very low dimer conversion rates after several freeze / thaw cycles. For example, the monomers of the antibody constructs are adjusted to a concentration of 250 μg / ml, for example, in a general-purpose formulation buffer, and subjected to three freeze / thaw cycles (freezing at -80°C for 30 minutes, followed by thawing at room temperature for 30 minutes), followed by high-speed SEC to determine the percentage of the initial monomeric antibody construct that has been converted to a dimeric antibody construct. Preferably, the percentage of dimers in the bispecific antibody construct is ≦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%, for example, after three freeze / thaw cycles.

[0222] The bispecific antibody constructs of the present invention 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. The thermal stability parameter in terms of the aggregation temperature of an antibody can be determined as follows: An antibody solution at a concentration of 250 μg / ml is transferred into 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, with the radius measurement being continuously acquired. The increase in radius, which indicates melting and aggregation of the protein, is used to calculate the aggregation temperature of the antibody.

[0223] Alternatively, melting temperature curves can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of antibody constructs. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. The energy uptake of samples containing the antibody constructs is recorded from 20°C to 90°C and compared to samples containing formulation buffer only. The antibody constructs are adjusted to a final concentration of 250 μg / ml, for example, in SEC running buffer. The overall temperature of the samples is increased stepwise to record each melting curve. The energy uptake of the samples and formulation buffer standards at each temperature T is recorded. The difference in energy uptake Cp (kcal / mole / °C) of the samples minus the standards is plotted against each temperature. The melting temperature is defined as the temperature at which energy uptake first reaches a maximum.

[0224] It is also envisaged that the target cell surface antigen x CD3 bispecific antibody constructs of the invention will have a turbidity (measured by OD340 after concentrating the purified monomeric antibody construct to 2.5 mg / ml and incubating overnight) of ≦0.2, preferably ≦0.15, more preferably ≦0.12, even more preferably ≦0.1, and most preferably ≦0.08.

[0225] In a further embodiment, the antibody construct according to the present invention is stable at physiological pH or slightly lower pH, i.e., about pH 7.4 to 6.0. The higher the tolerance of the antibody construct at non-physiological pH, e.g., about pH 6.0, the higher the recovery rate of the antibody construct eluted from the ion exchange column relative to the total amount of loaded protein. The recovery rate of the antibody construct from the ion (e.g., cation) exchange column at about pH 6.0 is preferably ≥ 30%, more preferably ≥ 40%, more preferably ≥ 50%, even more preferably ≥ 60%, even more preferably ≥ 70%, even more preferably ≥ 80%, even more preferably ≥ 90%, even more preferably ≥ 95%, and most preferably ≥ 99%.

[0226] It is further envisaged that the bispecific antibody constructs of the invention will exhibit therapeutic efficacy or anti-tumour activity, which can be assessed, for example, in the test disclosed in the Examples below in advanced stage human tumour xenograft models.

[0227] Those skilled in the art know how to vary or adapt certain parameters of this test, such as the number of tumor cells injected, the injection site, the number of transplanted human T cells, the amount of bispecific antibody construct administered, and the timeline, and still obtain meaningful and reproducible results. Preferably, the tumor growth inhibition T / C [%] is ≦70 or ≦60, more preferably ≦50 or ≦40, even more preferably ≦30 or ≦20, and most preferably ≦10 or ≦5, or even ≦2.5.

[0228] In a preferred embodiment of the antibody construct of the present invention, the antibody construct is a single chain antibody construct.

[0229] In a preferred embodiment of the antibody construct of the present invention, the third domain comprises, in order from amino to carboxyl: Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 Includes.

[0230] Also, in one embodiment of the present invention, the CH2 domain of one or preferably each (both) polypeptide monomer of the third domain comprises an intradomain cysteine ​​disulfide bridge. As known in the art, the term "cysteine ​​disulfide bridge" refers to a functional group having the general structure RSSR. This linkage, also known as an S-S bond or disulfide bridge, is obtained by coupling of two thiol groups of cysteine ​​residues. With regard to the antibody construct of the present invention, it is particularly preferred that the cysteines that form the cysteine ​​disulfide bridge in the mature antibody construct are introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0231] 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 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).

[0232] For example, it is believed that the preferred features of the antibody constructs of the present invention compared to bispecific hetero-Fc antibody constructs known in the art may relate, inter alia, to the introduction of the above-mentioned modifications in the CH2 domain. Thus, with respect to the constructs of the present invention, it is preferred that the CH2 domain within the third domain of the antibody construct of the present invention comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321 and / or the glycosylation site at Kabat position 314 is removed by an N314X substitution, preferably an N314G substitution, as described above.

[0233] In a further preferred embodiment of the invention, the CH2 domain within the third domain of an antibody construct of the invention comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 is eliminated by an N314G substitution.

[0234] In one embodiment, the present invention provides an antibody construct comprising: (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) An antibody construct is provided, wherein the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

[0235] Thus, the first and second domains may each be binding domains comprising two antibody variable domains, such as a VH and a VL domain. Examples of such binding domains comprising two antibody variable domains as described herein above include, for example, the Fv fragment, scFv fragment, or Fab fragment described herein above. Alternatively, either or both of the binding domains may comprise only a single variable domain. Examples of such single domain binding domains as described herein above include nanobodies or single variable domain antibodies comprising only one variable domain, which may be, for example, a VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0236] In a preferred embodiment of the antibody construct of the invention, the first and second domains are fused to the third domain via a peptide linker. Preferred peptide linkers are described herein above 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 equal to or greater than 1 (e.g., 2 or 3). A particularly preferred linker for fusion of the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0237] In a preferred embodiment, the antibody constructs of the invention comprise, in amino to carboxyl order: (a) First domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 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) 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) a second polypeptide monomer of the third domain The present invention is characterized by comprising:

[0238] In one embodiment of the present invention, the target cell surface antigen bound by the first domain is a tumor antigen, an antigen specific to an immune disorder, or a viral antigen. As used herein, the term "tumor antigen" can be understood as those antigens presented on tumor cells. These antigens can be presented on the cell surface with an extracellular portion, and often have both transmembrane and cytoplasmic portions of the molecule. These antigens may 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 common antigens are antigens presented by both tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or they may be accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. Non-limiting examples of tumor antigens as used herein are CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, CD20, CD70, BCMA, and PSMA.

[0239] Further target cell surface antigens specific for immunological disorders in the context of the present invention include, for example, TL1A and TNF-alpha. Preferably, said targets are addressed by the bispecific antibodies of the present invention, which are preferably full-length antibodies. In a highly preferred embodiment, the antibodies of the present invention are hetero-IgG antibodies.

[0240] In a preferred embodiment of the antibody construct of the invention, the tumor antigen is selected from the group consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, CD20, CD70, BCMA and PSMA.

[0241] In one aspect of the invention, the antibody construct comprises, in amino to carboxyl order: (a) SEQ ID NOs: 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, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 80, 81, 89, 90, 91, 92, 9 a first domain having an amino acid sequence selected from the group consisting of: 5, 131, 132, 133, 134, 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, and 181; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 189; (c) a second domain having 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 WO 2008 / 119567 or SEQ ID NO: 202; (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 a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24 of WO 2017 / 134140; (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 a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17 to 24 of WO 2017 / 134140;

[0242] In one aspect, the bispecific antibody construct of the invention binds to the surface antigens of the respective target cells: (a) SEQ ID NOs: 27, 28, 37-41; CD33 (b) each of SEQ ID NOs: 48 to 52; EGFRvIII (c) each of SEQ ID NOs: 59 to 64; MSLN (d) each of SEQ ID NOs: 71 to 82; CDH19 (e) each of SEQ ID NOs: 100 to 104; DLL3 (f) SEQ ID NOs: 7, 8, 17, 113 and 114; CD19 (g) each of SEQ ID NOs: 89 to 93; FLT3 (h) each of SEQ ID NOs: 121 to 125; CDH3 (i) each of SEQ ID NOs: 132 to 136; BCMA (j) each of SEQ ID NOs: 143 to 151, 158 to 166, and 173 to 181; PSMA (k) each of SEQ ID NOs: 212 and 213; MUC17 (l) each of SEQ ID NOs: 223, 224, 225, 236, and 237; and CLDN18 (m) each CD70 of SEQ ID NOs: 247 to 248 and characterized by having an amino acid sequence directed thereto.

[0243] The present invention further provides polynucleotides / nucleic acid molecules encoding the antibody constructs of the present invention. Polynucleotides are biological polymers composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (e.g., cDNA) and RNA (e.g., mRNA) are examples of polynucleotides with 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 or single-stranded, linear or circular. They are preferably contained within a vector contained within a host cell. The host cell is capable of expressing the antibody construct, for example, after transformation or transfection with the vector or polynucleotide of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a regulatory sequence.

[0244] The genetic code is a set of rules for translating information encoded in genetic material (nucleic acids) into proteins. Biological decoding in living cells is carried out by ribosomes, which transport amino acids and read mRNA three nucleotides at a time using tRNA molecules, which attach amino acids in the order specified by the mRNA. This code defines how triplet nucleotide sequences, called codons, specify the amino acid to be added next during protein synthesis. With some exceptions, each triplet codon in a nucleic acid sequence specifies a single amino acid. Because the majority of genes are coded using the exact same code, this particular code is often referred to as the canonical 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.

[0245] The present invention further provides a vector comprising the polynucleotide / nucleic acid molecule of the present invention. A vector is a nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into cells. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Genetically engineered vectors generally contain an origin of replication, a multiple cloning site, and a selection marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may contain additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically intended for the expression of transgenes in target cells and generally contain regulatory sequences.

[0246] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0247] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; 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 so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0248] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves creating transient pores or "holes" in the cell membrane to allow uptake of materials. Transfection can be achieved using calcium phosphate, by electroporation, by cell compression, or by mixing cationic lipids with substances to form liposomes, which fuse with the cell membrane and accumulate the cargo inside.

[0249] The term "transformation" is used to refer to the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells, including plant cells. Transformation is thus the genetic modification of bacteria or non-animal eukaryotic cells resulting from the direct uptake of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane and subsequent incorporation. Transformation can occur by artificial means. For transformation to occur, cells or bacteria must be in a competent state in which transformation can occur as a timed response to environmental conditions such as starvation and cell density.

[0250] The present invention further provides host cells transformed or transfected with the polynucleotide / nucleic acid molecule or vector of the present invention. As used herein, the term "host cell" or "recipient cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody construct of the present invention; and / or the antibody construct itself. Introduction of the respective substance into the cell is accomplished by transformation, transfection, etc. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Because certain modifications may occur in successive generations due to either spontaneous, accidental, or deliberate mutation, or due to environmental influences, such progeny may not, in fact, be completely identical (morphologically or in terms of genome or total DNA set) to the parent cell, but still be within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, and include, but are not limited to, 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.

[0251] The antibody constructs of the present invention can be produced in bacteria. After expression, the antibody constructs of the present invention can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, by affinity chromatography and / or size exclusion. Final purification can be carried out, for example, similar to the purification method for antibodies expressed in CHO cells.

[0252] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the antibody constructs of the present invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and useful in the present invention, such as Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 16045), and others. Kluyveromyces hosts such as Kluyveromyces spp. (36906), K. thermotolerans, and K. marxianus; Yarrowia spp. (EP 402226); Pichia pastoris (EP 183070); Candida spp.; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces occidentalis; Schwanniomyces, such as A. occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0253] Suitable host cells for expression of the glycosylated antibody constructs of the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as the viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0254] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins 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.

[0255] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney 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 carcinoma 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 (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0256] In a further embodiment, the present invention provides a method for the production of an antibody construct of the present invention, the method comprising culturing a host cell of the present invention under conditions that allow expression of the antibody construct of the present invention, and recovering the produced antibody construct from the culture.

[0257] As used herein, the term "culturing" refers to the maintenance, differentiation, growth, proliferation and / or propagation of cells in vitro under suitable conditions in a culture medium. The term "expression" includes any step involved in producing an antibody construct of the invention, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0258] When using recombinant techniques, antibody constructs can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the antibody construct is produced intracellularly, the first step is to remove particulate debris from 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 periplasmic space of Escherichia coli (E. coli). Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0259] The antibody constructs of the present invention prepared from host cells can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Depending on the antibody recovered, other protein purification techniques, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available. When the antibody construct of the present invention contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification.

[0260] Affinity chromatography is the preferred purification technique. The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0261] The present invention also provides pharmaceutical compositions comprising the antibody constructs of the present invention or antibody constructs produced according to the methods of the present invention. In the pharmaceutical compositions of the present invention, the homogeneity of the antibody constructs is preferably ≥80%, more preferably ≥81%, ≥82%, ≥83%, ≥84%, or ≥85%, even more preferably ≥86%, ≥87%, ≥88%, ≥89%, or ≥90%, even more preferably ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%, and most preferably ≥96%, ≥97%, ≥98%, or ≥99%.

[0262] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise one or more antibody constructs of the present invention, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Components of acceptable compositions are preferably non-toxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0263] The composition of the present invention may contain a pharmaceutically acceptable carrier. Generally, as used herein, "pharmaceutically acceptable carrier" refers to any aqueous or non-aqueous solution, sterile solution, solvent, buffer solution, such as phosphate-buffered saline (PBS) solution, water, suspension, emulsion such as oil / water emulsion, various types of wetting agents, liposomes, dispersion media and coatings that are suitable for pharmaceutical administration, especially 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.

[0264] Certain embodiments provide pharmaceutical compositions comprising an antibody construct of the invention and one or more additional excipients, such as those illustratively described in this section and elsewhere herein. Excipients can be used in the invention for a wide range of purposes, including methods of the invention to adjust the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or to improve efficacy and / or stabilize such formulations, as well as against degradation and damage due to stresses encountered during and after manufacture, transportation, storage, preparation prior to use, and administration.

[0265] In certain embodiments, pharmaceutical compositions may contain formulating materials intended to modify, sustain, or protect, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulating materials may include, but are not limited to, the following: charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine, such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine Antibacterial and antifungal agents Antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium bisulfite; buffers, buffer systems and buffering agents used to maintain the composition at physiological pH or slightly lower; examples of buffers are borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids, succinate, phosphate, and histidine; for example, Tris buffer at about pH 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, including water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media; · Biodegradable polymers such as polyester; Bulking agents such as mannitol or glycine; · Chelating agents such as ethylenediaminetetraacetic acid (EDTA); ·Isotonic and absorption retarding agents; complexing agents, e.g. caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin) · Injectables; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); 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 immunoglobulins, preferably of human origin; · Colouring and flavouring agents; Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate · Diluents; ·emulsifier; Hydrophilic polymers such as polyvinylpyrrolidone · Salt-forming counterions such as sodium; Preservatives, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like; examples are 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 cosolvents (such as glycerin, propylene glycol or polyethylene glycol); sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol; and polyhydric sugar alcohols; · suspending agents; surfactants or wetting agents, such as pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be detergents, preferably with a molecular weight of >1.2 KD, and / or polyethers, preferably with 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; ·Stability enhancers such as sucrose or sorbitol; isotonicity enhancing agents, such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol; Parenteral delivery vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils; Intravenous delivery vehicles, including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0266] It will be apparent to one skilled in the art that different components of a pharmaceutical composition (e.g., those listed above) may have different effects, for example, amino acids may act as buffers, stabilizers and / or antioxidants; mannitol may act as a bulking agent and / or tonicity enhancer; sodium chloride may act as a delivery vehicle and / or tonicity enhancer, etc.

[0267] It is contemplated that the compositions of the present invention may contain, in addition to the polypeptides of the present invention as defined herein, further biologically active agents depending on the intended use of the composition. Such agents may be drugs known in the art that act on the gastrointestinal system, drugs that act as cytostatics, drugs that prevent hyperuricemia, drugs that inhibit immune responses (e.g., corticosteroids), drugs that modulate inflammatory responses, and drugs and / or cytokines that act on the circulatory system. It is also contemplated that the antibody constructs of the present invention may be applied in combination therapy, i.e., in combination with another anti-cancer drug.

[0268] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vitro clearance rate of the antibody construct of the present invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the antibody construct of the present composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired degree of purity, optionally with a compounding agent (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Additionally, in certain embodiments, the antibody constructs of the present invention may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0269] When parenteral administration is intended, therapeutic compositions for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antibody construct of the present invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody construct of the present invention is formulated as a sterile, isotonic solution, appropriately preserved. In certain embodiments, the formulation may include a formulation of the desired molecule with an agent capable of providing controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can be delivered via depot injection. In certain embodiments, hyaluronic acid, which has the effect of enhancing duration in the circulation, may also be used. In certain embodiments, the desired antibody construct may be introduced using an implantable drug delivery device.

[0270] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody constructs of the invention in sustained- or controlled-delivery / release formulations. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US 93 / 00829, which describes controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. Sustained-release formulations may comprise semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices can include polyesters, hydrogels, and polylactides (disclosed in U.S. Pat. No. 3,773,919 and EP 058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Applications Nos. 036,676; 088,046 and 143,949, which are incorporated by reference.

[0271] The antibody constructs may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are described in Remington's Pharmaceutical Sciences, 16 th edition, Oslo, A. Ed. (1980).

[0272] Pharmaceutical compositions used for in vivo administration are generally provided as sterile preparations. Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or as a solution. Parenteral compositions are generally filled into a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0273] Another aspect of the present invention includes the self-buffering antibody constructs of the formulations of the invention that can be used as pharmaceutical compositions, as described in International Patent Application WO 06138181 A2 (PCT / US 2006 / 022599). Various descriptions are available for protein stabilization and formulation materials and methods useful in this regard, see, for example, 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), particularly with respect to protein pharmaceuticals and processes for veterinary and / or human medical use, see in particular the sections relating to excipients and processes similar to those for the self-buffering protein formulations of the present invention.

[0274] Salts may be used in certain embodiments of the present invention, for example, to adjust the ionic strength and / or tonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other components of the compositions according to the present invention. As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein, reducing the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the protein's denatured peptide bond (--CONH). Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0275] Ionic species vary significantly in their effects on proteins. Several taxonomic rankings of ions and their effects on proteins have been developed and can be used in formulating pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are commonly used to denature and / or solubilize proteins ("salting in"). The relative effects of an ion on "salting in" and "salting out" define the ion's position on the Hofmeister series.

[0276] Free amino acids can be used in the antibody constructs of the formulations of the present invention according to various embodiments as bulking agents, stabilizers, and antioxidants, as well as for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful for ensuring proper cake structure and properties during lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0277] Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as glycerol and propylene glycol, and for purposes of this discussion, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. 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 tonicity of formulations. Among the polyols useful in selected embodiments of the present invention is mannitol, which is commonly used in lyophilized formulations to ensure cake structural stability. Mannitol ensures cake structural stability. It is generally used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting tonicity and for protecting against freeze-thaw stress during transportation or bulk preparation in manufacturing processes. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not included among the preferred polyols for use in the present invention. In addition, sugars that form such reactive species, such as sucrose, are also not included among the preferred polyols of the present invention, as they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a cryoprotectant, and in this regard can be used in the present invention.

[0278] Embodiments of the antibody construct of the present invention further comprise a surfactant. Protein molecules can be prone to surface adsorption and denaturation and subsequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These adverse interactions are generally inversely proportional to protein concentration and are usually exacerbated by physical agitation, such as that encountered during product transportation and handling. Surfactants are traditionally used to prevent, minimize, or reduce surface adsorption. Surfactants useful 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 protein conformational stability. In this regard, the use of surfactants is protein-specific, as any given surfactant typically stabilizes some proteins and destabilizes others.

[0279] Polysorbates are prone to oxidative degradation and often contain sufficient peroxides as supplied to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should be used with caution and, when used, at the lowest possible concentrations. In this respect, polysorbates exemplify the general rule that excipients should be used at the lowest possible concentrations.

[0280] Embodiments of the antibody construct of the formulation of the present invention further comprise one or more antioxidants. Detrimental oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Particularly useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations of the present invention are preferably water-soluble and maintain activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention. Antioxidants can damage proteins. For example, reducing agents, such as glutathione, can disrupt intramolecular disulfide bonds, among other things. Therefore, antioxidants for use in the present invention are selected, inter alia, to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation.

[0281] The formulations of the present invention may contain metal ions, which are protein cofactors and are required to form protein coordination complexes, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins. Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 , Mn +2 and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2The aggregation can be destabilized by Al +3 It may be increased by ions.

[0282] Embodiments of the antibody construct formulations of the present invention further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations involving multiple withdrawals from the same container. Their primary function is to inhibit microbial growth and ensure product sterility over the shelf life or usage period of the formulation. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use with small molecule parenteral drugs, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single-use only. However, the possibility of multi-dose formulations offers the added benefits of patient convenience and increased marketability. Human growth hormone (hGH) is a good example, where the development of preserved formulations has led to the commercialization of more convenient multi-use injection pens. At least four such pen devices containing preserved formulations of hGH 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. During the formulation and development of preserved dosage forms, several aspects must be considered. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in the dosage form for a concentration range that confers antimicrobial efficacy without compromising protein stability.

[0283] As expected, developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing the associated stability risks. For liquid formulations, preservative effectiveness and stability should be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0284] The antibody constructs disclosed herein can also be formulated as immunoliposomes. "Liposomes" are small vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for drug delivery to mammals. The components of liposomes are generally arranged in a bilayer structure similar to the lipid arrangement of biological membranes. Liposomes containing antibody constructs are prepared by methods known in the art, e.g., as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 97 / 38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody constructs of the present invention can be conjugated to liposomes via a disulfide exchange reaction as described by Martin et al. J. Biol. Chem. 257:286-288 (1982). Optionally, a chemotherapeutic agent is contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

[0285] After the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0286] The biological activity of the pharmaceutical compositions defined herein can be determined, for example, by cytotoxicity assays as described in the Examples below, in WO 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 of therapy using the pharmaceutical compositions of the present invention, or in vivo efficacy, refers to the effectiveness of the composition for its intended purpose, i.e., its ability to cause its desired effect, i.e., the depletion of pathological cells, e.g., tumor cells. In vivo efficacy can be monitored by established standard methods for each disease entity, including, but not limited to, white blood cell counts, differential counts, fluorescence-activated cell sorting, and bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used.Additionally, computed tomography, x-ray, and nuclear magnetic resonance imaging (e.g., response assessment based on the 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] Apr;17(4):1244]), positron emission tomography scanning, white blood cell count, differential, fluorescence activated cell sorting, bone marrow aspiration, lymph node biopsy / histology and various lymphoma-specific clinical chemistry parameters (e.g., lactate dehydrogenase), and other established standard methods may be used.

[0287] Another major challenge in the development of drugs, such as the pharmaceutical compositions of the present invention, is the predictable modulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of a candidate drug can be established, i.e., a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given pathology. Pharmacokinetic parameters of a drug that affect a drug's ability to treat a certain disease include, but are not limited to, half-life, volume of distribution, hepatic first-pass metabolism, and degree of serum binding. The efficacy of a given drug can be influenced by each of the above parameters. A contemplated feature of the antibody constructs of the present invention provided by a particular FC format is that they include differences, for example, in pharmacokinetic behavior. The half-life extended targeting antibody constructs of the present invention preferably exhibit a surprisingly increased residence time in vivo compared to "canonical" non-HLE versions of the antibody construct.

[0288] "Half-life" refers to the time it takes for 50% of an administered drug to be eliminated through biological processes, such as metabolism, excretion, etc. "Hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" refers to the degree of retention of a drug across various compartments of the body, such as intracellular and extracellular spaces, tissues and organs, and the distribution of the drug within these compartments. "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins, such as albumin, resulting in a reduction or elimination of the drug's biological activity.

[0289] Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rate, onset of action, 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 from administration of a drug until it is detected and measurable in the blood or plasma. "Tmax" is the time thereafter to reach the maximum blood concentration of the drug, and "Cmax" is the maximum blood concentration achieved by a given drug. All parameters affect the time it takes for a drug to reach the blood or tissue concentration required for biological effect. Pharmacokinetic parameters of bispecific antibody constructs exhibiting cross-species specificity, which can be determined in preclinical animal studies in non-chimpanzee primates as outlined above, can be determined, for example, by cytotoxicity assays as described in Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12).

[0290] In a preferred embodiment of the present invention, the pharmaceutical composition is stable for at least 4 weeks at about -20°C. As is evident from the accompanying examples, the quality of the antibody constructs of the present invention relative to the quality of corresponding state-of-the-art antibody constructs can be tested using different systems. These tests are understood to be in accordance with the "ICH Harmonized 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-indicating profile that allows reliable detection of changes in the identity, purity, and potency 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 more than one method, and the resulting purity value is method-dependent. For stability testing purposes, purity tests should be combined with methods for determining degradation products.

[0291] The quality of a pharmaceutical composition comprising an antibody construct of the present invention can be assessed, for example, by analyzing the content of soluble aggregates in solution (HMWS by size exclusion). Stability at about -20°C for at least 4 weeks is characterized by a content of less than 1.5% HMWS, preferably less than 1% HMWS.

[0292] A preferred method for analyzing product quality herein is size-exclusion high-performance liquid chromatography (SE-HPLC). SE-HPLC is typically performed using a size-exclusion column and a UHPLC system, such as a Waters BEH200 size-exclusion column (4.6 × 150 mm, 1.7 μm) and a Waters UHPLC system. Protein samples were injected directly and separated isocratically using, for example, a phosphate buffer containing NaCl salt (the mobile phase was 100 mM sodium phosphate, pH 6.8, 250 mM NaCl) at a flow rate of, for example, 0.4 mL / min, and the eluate was monitored by UV absorbance at 280 nm. Typically, approximately 6 μg of sample was loaded.

[0293] Before the CM process is initiated, a vial containing CHO cells expressing the bispecific antibody construct is typically thawed. During scale-up, the cells are resuspended in fresh selective growth medium at a target viable cell density (VCD). The culture volume is continuously increased in shake flasks or bioreactors to ultimately generate a sufficient amount of cells to seed a perfusion production bioreactor (e.g., at a scale of 10 L or 50 L or greater).

[0294] Once the cells are inoculated into the production bioreactor at a concentration range as specified herein, there is an initial cell growth phase for several days, typically about 7-28 days, to increase the cell density and biomass to the desired set point as described herein and as measured by a dielectric constant probe (Hamilton Bonaduz AG, Switzerland). The production bioreactor is controlled at a desired pH, typically about 6-7.4, e.g., pH 6.85, dissolved oxygen, e.g., 64 mm Hg, and about 36°C. Perfusion culture is initiated after several days of the cell growth phase, typically on day 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably on day 4, using a filter such as a polyethersulfone 0.2-μm filter (e.g., GE Healthcare, Pittsburg, PA) and an alternating tangential flow (ATF) filtration system (e.g., Refine Technologies, Hanover, NJ) with a suitable chemically defined perfusion medium at a VVD perfusion rate as described herein, e.g., 0.4 bioreactor VVD. The perfusion rate is usually gradually increased, e.g., from 0.4 VVD on day 4 to 2 VVD on day 12. Once the biomass set point is reached on the last day of the stepwise VVD increase, the cell culture temperature is typically lowered, e.g., to 33.5°C, HCCF harvesting is initiated (i.e., cell-free permeate containing the bispecific antibody construct), and the perfusion culture is continued for a period as described herein, e.g., at least 7, 14, 28, or 40 additional days, preferably at least 28 days, by supplying at the set perfusion rate, typically the highest VVD permeate rate the stepwise increase leads to (i.e., steady-state cell specific permeation rate, CSPR, e.g., 0.02-0.03 nL / cell-day), and withdrawing excess cells to maintain the desired biomass set point. Cell density (CDV, e.g., measured by Nova Biomedical, Waltham, MA), metabolites (e.g., measured by NovaFlex, Nova Biomedical, Waltham, MA), and permeate titer (measured by HPLC analysis) are typically measured throughout the culture period.The HCCF is preferably collected continuously at room temperature or at increments of, for example, 6, 12, 24, 48, 72, 96, 120, or 144 hours and processed to Protein-L capture chromatography. For example, the eluates from Protein-L on days 26, 27, 34, and 40 are analyzed for product quality attributes and process-related impurities using analytical cation exchange chromatography (CEX-HPLC), peptide mapping, and / or HCP ELISA.

[0295] Tryptic peptide mapping of chemical modifications Protein samples of bispecific antibody constructs are digested using a filter-based method, for example, using a Millipore Microcon 30K device. The protein sample is loaded onto a filter and centrifuged to remove the sample matrix, followed by denaturation in, for example, 6 M guanidine hydrochloride (GuHCl) (e.g., Thermo Fisher Scientific, Rockford, IL) buffer containing methionine, reduction with, for example, 500 mM dithiothreitol (DTT) (e.g., Sigma-Aldrich, St. Louis, MO) for 30 minutes at 37°C, and then alkylation with, for example, 500 mM iodoacetic acid (IAA) (e.g., Sigma-Aldrich, St. Louis, MO) for 20 minutes at room temperature in the dark. Unreacted IAA is quenched by the addition of DTT. All of the above steps were performed on the filter. The sample is then buffer exchanged into a digestion buffer (e.g., 50 mM Tris, pH 7.8, containing methionine) by centrifugation to remove residual DTT and IAA. Trypsin digestion is performed on the filter using an enzyme-to-protein ratio of 1:20 (w / w), for example, at 37°C for 1 hour. The digestion mixture is recovered by centrifugation and subsequently quenched by adding, for example, 8 M GuHCl in acetate buffer, pH 4.7.

[0296] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using an ultra-performance liquid chromatography (UPLC) system, such as the Thermo U-3000, directly coupled to a mass spectrometer, such as the Thermo Scientific Q-Exactive. Protein digests were separated by reversed phase chromatography using an Agilent Zorbax C18 RR HD column (2.1 x 150 mm, 1.8 μm) with the column temperature maintained at 50 °C. Mobile phase A consisted of 0.020% (v / v) formic acid (FA) in water, and mobile phase B was 0.018% (v / v) FA in acetonitrile (I). Approximately 5 μg of digested bispecific antibody construct was injected onto the column. Peptides were separated using a gradient (e.g., 0.5–36% B over 145 min) at a flow rate of, for example, 0.2 mL / min. Eluted peptides were monitored by MS.

[0297] For peptide identification and modification analysis, data-dependent tandem MS (MS / MS) experiments are typically utilized. A full scan is typically acquired, for example, from 200 to 2000 m / z in positive ion mode, followed by, for example, six data-dependent MS / MS scans to identify the peptide sequence. Quantification is based on selected ion monitoring mass spectrometry data using the formula below:

number

[0298] Host Cell Protein (HCP) ELISA Microtiter plates are coated with rabbit anti-HCP immunoglobulin G (IgG) (Amgen, in-house antibody). After washing and blocking, test samples, controls, and HCP calibration standards are added to the plate and incubated. Unbound proteins are washed from the plate, and pooled rabbit anti-HCP IgG-biotin (Amgen, in-house antibody) is added to the plate and incubated. After further washing, streptavidin™ horseradish peroxidase conjugate (HRP-conjugate) (e.g., Amersham-GE, Buckinghamshire, UK) is added to the plate and incubated. The plate is washed a final time, and the chromogenic substrate tetramethylbenzidine (TMB) (e.g., Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added to the plate. Color development is stopped with 1 M phosphoric acid, and the optical density is measured in a spectrophotometer.

[0299] A preferred formulation for the antibody construct as a pharmaceutical composition may, for example, include the components of the formulation as follows: ·formulation: Potassium phosphate pH 6.0, L-arginine hydrochloride, trehalose, polysorbate 80

[0300] In general, antibody constructs provided in a specific FC format according to the present invention are expected to be more stable against a wide range of stress conditions, such as temperature and light stress, compared to both antibody constructs provided with different HLE formats and antibody constructs without any HLE format (e.g., "canonical" antibody constructs). The aforementioned 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 will make the antibody construct safer in clinical practice, as fewer degradation products will be produced. Consequently, the aforementioned improved stability means improved safety.

[0301] One embodiment provides an antibody construct of the invention or an antibody construct produced according to a method of the invention for use in the prevention, treatment or amelioration of a proliferative disease, a neoplastic disease, a viral disease or an immune disorder.

[0302] The formulations described herein are useful as pharmaceutical compositions for treating, ameliorating, and / or preventing the pathological medical conditions described herein in a patient in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the formulations to the body, isolated tissues, or cells of a patient with a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, treat, relieve, alleviate, alter, correct, ameliorate, improve, or affect the disease, symptom of a disease, or predisposition to a disease.

[0303] As used herein, the term "amelioration" refers to any improvement in the disease state of a patient having a tumor or cancer or metastatic cancer as defined herein below, by administration of an antibody construct according to the present invention to a subject in need thereof. Such improvement may be seen as a slowing or halting of the progression of the patient's tumor or cancer or metastatic cancer. As used herein, the term "prevention" refers to the avoidance of the onset or recurrence of a patient having a tumor or cancer or metastatic cancer as defined herein below, by administration of an antibody construct according to the present invention to a subject in need thereof.

[0304] The term "disease" refers to any condition that would benefit from treatment with the antibody constructs or pharmaceutical compositions described herein, including chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question.

[0305] A "neoplasm" is an abnormal growth of tissue, usually, although not necessarily, forming a mass. When it forms a mass, it is commonly called a "tumor." A neoplasm or tumor can be benign, potentially malignant (precancerous), or malignant. Malignant neoplasms are commonly called cancers. They usually invade and destroy surrounding tissues and can form metastases, i.e., they spread to other parts, tissues, or organs of the body. Thus, the term "metastatic cancer" includes metastases to tissues or organs other than that of the primary tumor. Lymphomas and leukemias are lymphatic neoplasms. For the purposes of the present invention, they are also encompassed by the terms "tumor" or "cancer."

[0306] The term "viral disease" refers to a disease that is the result of a viral infection in a subject.

[0307] As used herein, the term "immune disorder" refers to immune disorders such as autoimmune diseases, hypersensitivity disorders, and immune deficiencies in accordance with the general definition of the term.

[0308] In one embodiment, the present invention provides a method for the treatment or amelioration of a proliferative disease, a neoplastic disease, a viral disease or an immune disorder, comprising the step of administering to a subject in need thereof an antibody construct of the present invention or an antibody construct produced according to a method of the present invention.

[0309] The term "subject in need" or "subject in need of treatment" includes subjects already with the disorder as well as subjects in which the disorder is to be prevented. A subject in need or a "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0310] The antibody constructs of the present invention will generally be designed for a particular route and method of administration, a particular dosage and frequency, and a particular treatment of a particular disease, particularly in the areas of bioavailability and duration. The materials of the composition are preferably formulated in concentrations that are acceptable to the site of administration.

[0311] Thus, formulations and compositions may be designed for delivery by any suitable route of administration in accordance with the present invention. ·Topical route (e.g., on the skin, inhalation, nose, eyes, pinna / ear, vagina, mucous membranes); Enteral routes (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, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal) Including, but not limited to:

[0312] The pharmaceutical compositions and antibody constructs of the invention are particularly useful for parenteral administration, e.g., subcutaneous or intravenous delivery, e.g., by injection, e.g., bolus injection, or by infusion, e.g., continuous infusion. The pharmaceutical composition may 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.

[0313] In particular, the present invention provides for uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e., continuous, administration can be achieved by a miniature pump system worn by the patient to regulate the influx of a therapeutic agent into the patient's body. Pharmaceutical compositions comprising antibody constructs of the present invention can be administered using such pump systems. Such pump systems are generally known in the art and typically rely on periodic replacement of a cartridge containing the therapeutic agent to be infused. When replacing the cartridge in such pump systems, a temporary interruption in the otherwise uninterrupted flow of therapeutic agent into the patient's body may result. Even in such cases, the administration step before cartridge replacement and the administration step after cartridge replacement would still be considered within the meaning of the pharmaceutical means and methods of the present invention, together constituting the "uninterrupted administration" of such therapeutic agent.

[0314] Continuous or uninterrupted administration of the antibody construct of the present invention can be intravenous or subcutaneous administration using a fluid delivery device or miniature pump system, which includes a fluid pumping mechanism for pumping fluid from a reservoir and a drive mechanism for driving the pumping mechanism. A pump system for subcutaneous administration can include a needle or cannula for penetrating the patient's skin and delivering the suitable composition into the patient's body. The pump system can be fixed or attached directly to the patient's skin, whether it be a vein, artery, or blood vessel, allowing for direct contact between the pump system and the patient's skin. This pump system can be attached to the patient's skin for 24 hours to several days. In some cases, the pump system can be a miniature pump system with a small reservoir volume. As a non-limiting example, the reservoir volume for the suitable pharmaceutical composition to be administered can be 0.1 to 50 ml.

[0315] Continuous administration can also be transdermal, by means of a patch that is attached to the skin and replaced from time to time.Those skilled in the art are aware of suitable patch systems for drug delivery for this purpose.It should be noted that transdermal administration is particularly suitable for uninterrupted administration, since it has the advantage that, for example, a new second patch can be attached to the skin surface directly adjacent to the first used patch immediately before removing the first used patch, and the replacement of the first used patch can be completed at the same time.There is no problem of interruption of inflow or battery failure.

[0316] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with an appropriate liquid prior to administration, for example, bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation the protein was in before lyophilization.

[0317] The compositions of the present invention can be administered to a subject at a suitable dose, which can be determined, for example, by a dose-escalation study in which the antibody constructs of the present invention exhibiting cross-species specificity described herein are administered to non-chimpanzee primates, such as macaques, in increasing doses. As described above, the antibody constructs of the present invention exhibiting cross-species specificity described herein have the advantage that they can be used in the same form in preclinical studies in non-chimpanzee primates and as drugs in humans. The dosing regimen will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage administered to a given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly.

[0318] The term "effective dose" or "effective administration amount" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The amount or dosage effective for this use will depend on the condition (indication) being treated, the antibody construct being delivered, the nature and purpose of the treatment, the severity of the disease, previous treatments, the patient's medical history and responsiveness to the therapeutic agent, the route of administration, the size (weight, body surface area, or organ size) and / or condition (age and general health) of the patient, and the general status of the patient's own immune system. The appropriate dosage can be administered to the patient in a single administration or multiple administrations, and can be adjusted according to the judgment of the attending physician to obtain the optimal therapeutic effect.

[0319] Typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In certain embodiments, dosages can range from 1.0 μg / kg up to about 20 mg / kg, optionally 10 μg / kg up to about 10 mg / kg, or 100 μg / kg up to about 5 mg / kg.

[0320] A therapeutically effective amount of an antibody construct of the invention preferably reduces the severity of disease symptoms, increases the frequency or duration of symptom-free periods, or prevents impairment or disability due to disease affliction. With respect to treating antigen-expressing tumors of target cells, a therapeutically effective amount of an antibody construct of the invention, e.g., an anti-target cell antigen / anti-CD3 antibody construct, 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 a compound to inhibit tumor growth can be assessed in animal models predictive of efficacy.

[0321] The pharmaceutical composition can be administered in a single treatment or, if necessary, in combination with additional treatments, such as anti-cancer therapies, e.g., other proteinaceous and non-proteinaceous drugs, which can be administered simultaneously with the composition comprising the antibody construct of the present invention as defined herein, or can be administered separately at predetermined time intervals and dosages before or after administration of said antibody construct.

[0322] As used herein, the term "effective and non-toxic dose" refers to a tolerated dose of an antibody construct of the present invention that is sufficient to result in depletion of pathological cells, tumor elimination, tumor regression, or disease stabilization without or essentially without significant toxic effects. Such an effective and non-toxic dose can be determined, for example, by dose escalation studies as described in the art, and should be below the dose that induces serious adverse side effects (dose-limiting toxicity, DLT).

[0323] As used herein, the term "toxicity" refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These side effects may refer to a lack of systemic drug tolerance and / or a lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0324] 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 tolerance) and during longer drug application periods. "Safety," "in vivo safety," or "tolerability" can be assessed, for example, periodically during treatment and follow-up. Measurements include clinical evaluations, such as organ findings and screening for laboratory abnormalities. Clinical evaluations can be performed, and deviations from normal findings can be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings can include, for example, criteria set forth in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE), such as allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation, etc. Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile, and urinalysis, as well as tests of other body fluids, such as serum, plasma, lymph, or spinal fluid. Thus, safety can be assessed, for example, by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI)), other measurements using technical devices (i.e. electrocardiogram), vital signs, measuring laboratory parameters and recording adverse events. For example, in the uses and methods according to the invention, adverse events in non-chimpanzee primates can be tested by histopathological and / or histochemical methods.

[0325] The above terms are also referenced, for example, in Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6 of July 16, 1997; ICH Harmonized Tripartite Guideline; ICH Steering Committee meeting.

[0326] Finally, the present invention provides kits comprising the antibody construct of the invention, the antibody construct produced according to the method of the invention, the pharmaceutical composition of the invention, the polynucleotide of the invention, the vector of the invention and / or the host cell of the invention.

[0327] In the context of the present invention, the term "kit" refers to two or more components packaged together in a container, vessel, or other arrangement, one of which corresponds to an antibody construct, pharmaceutical composition, vector, or host cell of the present invention. A kit can therefore be described as a set of products and / or implements sufficient to achieve a particular purpose that can be sold as a single item.

[0328] The kit may comprise one or more containers (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing an antibody construct or pharmaceutical composition of the invention in a dosage amount suitable for administration (see above). The kit may further comprise instructions for use (e.g., in the form of a leaflet or instruction manual), a means for administering the antibody construct of the invention, e.g., a syringe, pump, infuser, etc., a means for reconstituting the antibody construct of the invention, and / or a means for diluting the antibody construct of the invention.

[0329] The present invention also provides kits for single-dose administration units. The kits of the present invention may also include a first container containing a dried / lyophilized antibody construct 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).

[0330] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "reagent" includes one or more of such different reagents, and a reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified for or substituted for the methods described herein.

[0331] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0332] The term "and / or", wherever used in this specification, includes the meanings "and", "or" and "any other combination of the elements connected by said term".

[0333] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range, although the term also includes specific numbers, for example, about 20 includes 20.

[0334] The terms "less than" or "greater than" are inclusive of specific numbers. For example, less than 20 means less than or equal to. Similarly, greater than or greater than means greater than or equal to, or greater than or equal to, respectively.

[0335] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" can also be replaced with the terms "containing" or "including," or sometimes the term "having," when used herein.

[0336] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0337] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with one of the other two terms.

[0338] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0339] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), cited throughout the text of this specification, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will take precedence over any such material.

[0340] A better understanding of the present invention and its advantages will be obtained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0341] Example 1: Continuous manufacturing: Automated biomass-based feeding for improved productivity and robustness For this process, a commercial-scale 50 L single-use bioreactor was used. The overall duration was 40 days of continuous bioreactor operation and 28 days of continuous harvest. Automated biomass control according to the present invention was applied. The test antibody construct was the CD19xCD3 bispecific T cell engager molecule (SEQ ID NO: 17). The expressing cells were CHO cells.

[0342] At bench scale (1.5 L working volume), three experiments were performed to test biomass-based feeding.

[0343] In the first experiment, manual PCV-based feeding [volume / day / % PCV] was tested during the production phase of a continuous perfusion process. After day 23, the PCV setpoint was manually increased with a corresponding increase in feeding rate (based on BSPR). The BSPR setpoint was 0.078 l / day from days 23 to 33. The control process had a manual time-based feeding rate [volume / day] and a fixed PCV. As a result, a PCV of 50% was achieved with high viability (Figures 2-3) and a metabolite profile similar to the control.

[0344] In a second experiment, automated permittivity-based feeding was tested during the growth phase of a continuous perfusion process. The test condition had a BSPR of 0.04 cm / pF·day on days 4–10 and 0.03 cm / pF·day on days 11–14. The control process had a manual time-based feeding rate [volume / day] during the growth phase. The test condition had similar cell growth and metabolite profiles to the control (Figures 4 and 5).

[0345] In the third experiment, three levels of biomass were tested: 19%, 23%, and 27% packed cell volume (PCV). During the continuous harvest period, the control process (constant feed rate) was run for 14 days, and the investigational automated biomass-based feed was run for 6 days. No new hardware was required, but the integration of two control loops for level control and biomass control was required. Biomass specific perfusion rate (BSPR) was calculated from the control setpoint.

[0346] As a result, better biomass control was observed under automated biomass-based feeding compared to control feeding (see Figure 6). Similar osmolality and lactate values ​​were observed across biomass levels (see Figure 7). Titers were comparable to the control strategy and were higher at higher biomass levels (see Figure 7). Even when biomass and feed rates were controlled within range, small increases in BSPR can increase titers. Advantageously, viability remained high (>89%) in the bioreactor under all conditions.

[0347] Example 2 The CD70xCD3 bispecific molecule (SEQ ID NO: 248) was produced under biomass-based control. The process was a 15-28 day extended perfusion process (continuous manufacturing) using ATF technology. The cell culture was operated in a non-steady-state mode.

[0348] Automated permittivity-based feeding was tested at three CSPR levels in both the growth phase (0.02, 0.03, and 0.065 cm / pF / day) and production phase (0.01, 0.017, and 0.035 cm / pF / day) and compared to a continuous production control. Automated biomass-based feeding (circles) with similar CSPR performed similarly to the control (triangles). At low CSPR (dashes), productivity was high and survival was low during the production phase. Conversely, at high CSPR (crosses), productivity was low and survival was high (see Figure 9).

[0349] [Table 4]

[0350] Example 3 A PD1 IL21 mutein bispecific molecule was produced. The process was a 15-day perfusion process using ATF technology. A single CSPR value was tested in duplicate during the growth and production phases. A high CSPR (0.12) was tested on days 3-8, and a low CSPR (0.03) was tested on days 9-15 (see Figure 10). Titers were lower under automated feeding compared to the control, but productivity was similar. This is likely due to lower cell growth under automated feeding conditions (see Figure 10). Optimizing the CSPR amount and timing increased VCD and titers to approximately 0.03-0.05 cm / pF / day during the growth phase and 0.01-0.025 cm / pF / day during the production phase.

[0351] [Table 5]

[0352] Example 4 PD1 mAb was produced. The process was a 15-day perfusion process using ATF technology. One CSPR value was tested in duplicate during the growth and production phases. A high CSPR (0.08) was tested on days 3-8, and a low CSPR (0.015) was tested on days 9-15. Titers were slightly lower in the automated feeding compared to the control, but productivity was higher (see Figure 10). This is likely due to lower VCD in the automated feeding conditions. Higher productivity is expected to be associated with lower viability.

[0353] [Table 6]

Claims

1. 1. An upstream manufacturing process for the production of an antibody product applying automated measurement and regulation of perfusion flow in a perfusion bioreactor, comprising: (i) providing a liquid cell culture medium comprising at least one mammalian cell culture into the perfusion bioreactor, wherein the mammalian cell culture is capable of expressing the antibody product, and the cells have a concentration (viable cell density, VCD) of at least 1 x 10 cells / mL at the time of inoculation in the perfusion bioreactor; (ii) providing a first control loop for measuring and regulating medium level in the bioreactor, the first control loop including a level probe that measures the medium level in the bioreactor relative to a set point, a permeate pump calibrated to measure permeate volume (volume per time), and level control means receiving inputs from the level probe and the permeate pump that can correct the medium feed rate to the bioreactor in response to a medium pump (feed pump) in response to inputs from the level probe and the permeate probe, or that can correct the output rate from the bioreactor in response to inputs from the level probe and the medium probe, wherein measurements of the medium level in the bioreactor are taken at predetermined fixed time intervals; (iii) providing a second control loop for measuring and regulating biomass in the bioreactor, the second control loop comprising a dielectric or Raman probe, preferably a dielectric probe, in the bioreactor for measuring the biomass, and biomass control means receiving input from the biomass dielectric or Raman probe capable of correcting discharge from the bioreactor in response to a discharge pump depending on the input, wherein measurements of the biomass in the bioreactor are taken at predetermined fixed time intervals; (iv) providing integrated first and second control loops by connecting said biomass control means and said level control means to an integrated unit, said integrated unit being capable of performing an automated perfusion rate calculation, said perfusion rate being a function of said biomass value, preferably according to the formula Perfusion rate (mL / min) = function of biomass values ​​(dielectric constant, PCV, VCD, spectroscopic measurements) and / or Perfusion rate [mL / min] = Perfusion rate based on dielectric constant (constant) [cm / pF / d] × Dielectric constant value [pF / cm] wherein the constant is the perfusion rate [1 / d] divided by the dielectric constant [pF / cm], and the dielectric constant value is 0.5 to 120 pF / cm during a first period (growth phase) during which biomass in the bioreactor increases to about a predetermined biomass set point and / or 25 to 100 pF / cm during a second period (production phase) of biomass stabilization after reaching the predetermined biomass set point; (v) automatically correcting or maintaining the perfusion rate by the integrated unit, wherein the integrated unit sends a signal to the permeate pump and / or the medium pump to increase or decrease the pumping rate, respectively, depending on the biomass measured at predetermined fixed time intervals. A process involving:

2. 2. The process according to claim 1, wherein the upstream manufacturing process is a discontinuous manufacturing process, preferably a perfusion process and / or a fed-batch process, or a continuous manufacturing process, preferably a continuous perfusion process.

3. 2. The process of claim 1, wherein in step (i), the cells have a concentration of at least 7 x 10^5 cells / mL at the time of inoculation in the bioreactor.

4. 2. The process of claim 1, wherein in step (iv), the biomass set point is equal to a VCD of at least 30 x 10^6 cells / mL, preferably 30 x 10^6 cells / mL, if the manufacturing process is a discontinuous process, and equal to a VCD of 65 x 10^6 cells / mL if the manufacturing process is a continuous manufacturing process.

5. 2. The process of claim 1, wherein in step (iv), the growth of the cell culture occurs for at least 4 days, preferably at least 7 days, more preferably at least 12 or 14 days.

6. 2. The process according to claim 1, wherein in step (ii), the predetermined fixed time interval corresponds to at most 1 minute, preferably 30 seconds, more preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 seconds, preferably 1 second.

7. 2. The process according to claim 1, wherein in step (iii), the predetermined fixed time interval corresponds to at most 1 minute, preferably 30 seconds, more preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 seconds, preferably 1 second.

8. 2. The process according to claim 1, wherein in step (v), the predetermined fixed time interval corresponds to at most 1 minute, preferably 30 seconds, more preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 seconds, preferably 1 second.

9. 2. The process of claim 1, wherein the dielectric constant during the growth phase is between 0.70 and 120 pF / cm, preferably between 0.73 and 70.7 pF / cm, more preferably between 1 and 20 pF / cm or between 100 and 117 pF / cm, preferably when the manufacturing process is a continuous manufacturing process.

10. 2. The process of claim 1, wherein the specific cell perfusion rate based on the dielectric constant in continuous production is preferably 0.01-0.049 cm / pF / d, preferably 0.015-0.04 cm / pF / d, more preferably 0.02-0.04 cm / pF / d, most preferably 0.0266-0.04 cm / pF / d in the growth phase, or the specific cell perfusion rate based on the dielectric constant in case of discontinuous production is up to 0.2 cm / pF / d, more preferably up to 0.13 cm / pF / d.

11. 2. The process of claim 1, wherein the applied perfusion rate corresponds to a CSPR of 0.01-0.1 nL / cell / d during the growth phase, preferably 0.02-0.08 nL / cell / d, more preferably 0.027-0.076 nL / cell / d during the growth phase.

12. 2. The process of claim 1, wherein the dielectric constant in production is between 55 and 85 pF / cm, preferably between 60 and 75 pF / cm, more preferably between 62 and 73 pF / cm.

13. 2. The process of claim 1, wherein the dielectric constant-based specific cell perfusion rate is 0.01-0.04 cm / pF / d, preferably 0.01-0.035 cm / pF / d, preferably 0.01-0.0266 cm / pF / d during the production phase.

14. 2. The process according to claim 1, wherein the applied perfusion rate corresponds to a CSPR of 0.01 to 0.49 nL / cell / d, preferably 0.015 to 0.04 nL / cell / d, particularly preferably 0.023 to 0.035 nL / cell / d during the production phase.

15. 2. The process of claim 1, wherein the production period is at least 14 days, preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 days, wherein the production process is a continuous manufacturing process, or at least 3 days, preferably 4 or 5 days, wherein the production process is a discontinuous manufacturing process.

16. The process of claim 1, wherein the antibody product is a full-length antibody, such as a monoclonal antibody, preferably directed against PD-1, or a non-full-length molecule.

17. 17. The process according to claim 16, wherein the antibody product is preferably bispecific, i.e. preferably a full length antibody or a molecule based on a full length antibody or a fragment thereof that binds to a target and / or an effector cell, respectively.

18. 18. The process of claim 17, wherein the bispecific antibody product is a fusion protein, preferably an anti-PD-1 mAb / IL-21 mutein fusion protein.

19. 18. The process of claim 17, wherein the antibody product is a bispecific, non-full-length molecule comprising a first binding domain and a second binding domain that bind to a target and an effector molecule, respectively.

20. 18. The process of claim 17, wherein the bispecific molecule is a bispecific T cell engager molecule.

21. 21. The process of claim 20, wherein the bispecific molecule comprises a half-life extending moiety preferably selected from human serum albumin (HAS), an HAS binding domain, a heterologous Fc domain or an Fc-based half-life extending moiety derived from an IgG antibody, most preferably an scFc half-life extending moiety.

22. 20. The process of claim 19, wherein the first binding domain of the bispecific antibody construct binds to at least one target cell surface antigen selected from the group consisting of CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, EpCAM, CD70, MUC17, CLDN18, BCMA, and PSMA.

23. 20. The process of claim 19, wherein the second binding domain of the bispecific antibody product binds to CD3.

24. the first binding domain comprises: (a) CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6; (b) CDR-H1 as set forth in SEQ ID NO:29, CDR-H2 as set forth in SEQ ID NO:30, CDR-H3 as set forth in SEQ ID NO:31, CDR-L1 as set forth in SEQ ID NO:34, CDR-L2 as set forth in SEQ ID NO:35, and CDR-L3 as set forth in SEQ ID NO:36; (c) CDR-H1 as set forth in SEQ ID NO: 42, CDR-H2 as set forth in SEQ ID NO: 43, CDR-H3 as set forth in SEQ ID NO: 44, CDR-L1 as set forth in SEQ ID NO: 45, CDR-L2 as set forth in SEQ ID NO: 46, and CDR-L3 as set forth in SEQ ID NO: 47; (d) CDR-H1 as set forth in SEQ ID NO:53, CDR-H2 as set forth in SEQ ID NO:54, CDR-H3 as set forth in SEQ ID NO:55, CDR-L1 as set forth in SEQ ID NO:56, CDR-L2 as set forth in SEQ ID NO:57, and CDR-L3 as set forth in SEQ ID NO:58; (e) CDR-H1 as set forth in SEQ ID NO: 65, CDR-H2 as set forth in SEQ ID NO: 66, CDR-H3 as set forth in SEQ ID NO: 67, CDR-L1 as set forth in SEQ ID NO: 68, CDR-L2 as set forth in SEQ ID NO: 69, and CDR-L3 as set forth in SEQ ID NO: 70; (f) CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, and CDR-L3 as set forth in SEQ ID NO: 88; (g) CDR-H1 as set forth in SEQ ID NO: 94, CDR-H2 as set forth in SEQ ID NO: 95, CDR-H3 as set forth in SEQ ID NO: 96, CDR-L1 as set forth in SEQ ID NO: 97, CDR-L2 as set forth in SEQ ID NO: 98, and CDR-L3 as set forth in SEQ ID NO: 99; (h) CDR-H1 as set forth in SEQ ID NO: 105, CDR-H2 as set forth in SEQ ID NO: 106, CDR-H3 as set forth in SEQ ID NO: 107, CDR-L1 as set forth in SEQ ID NO: 109, CDR-L2 as set forth in SEQ ID NO: 110, and CDR-L3 as set forth in SEQ ID NO: 111; (i) CDR-H1 as set forth in SEQ ID NO: 115, CDR-H2 as set forth in SEQ ID NO: 116, CDR-H3 as set forth in SEQ ID NO: 117, CDR-L1 as set forth in SEQ ID NO: 118, CDR-L2 as set forth in SEQ ID NO: 119, and CDR-L3 as set forth in SEQ ID NO: 120; (j) CDR-H1 as set forth in SEQ ID NO: 126, CDR-H2 as set forth in SEQ ID NO: 127, CDR-H3 as set forth in SEQ ID NO: 128, CDR-L1 as set forth in SEQ ID NO: 129, CDR-L2 as set forth in SEQ ID NO: 130, and CDR-L3 as set forth in SEQ ID NO: 131; (k) CDR-H1 as set forth in SEQ ID NO: 137, CDR-H2 as set forth in SEQ ID NO: 138, CDR-H3 as set forth in SEQ ID NO: 139, CDR-L1 as set forth in SEQ ID NO: 140, CDR-L2 as set forth in SEQ ID NO: 141, and CDR-L3 as set forth in SEQ ID NO: 142; (l) CDR-H1 as set forth in SEQ ID NO: 152, CDR-H2 as set forth in SEQ ID NO: 153, CDR-H3 as set forth in SEQ ID NO: 154, CDR-L1 as set forth in SEQ ID NO: 155, CDR-L2 as set forth in SEQ ID NO: 156, and CDR-L3 as set forth in SEQ ID NO: 157; (m) CDR-H1 as set forth in SEQ ID NO: 167, CDR-H2 as set forth in SEQ ID NO: 168, CDR-H3 as set forth in SEQ ID NO: 169, CDR-L1 as set forth in SEQ ID NO: 170, CDR-L2 as set forth in SEQ ID NO: 171, and CDR-L3 as set forth in SEQ ID NO: 172; (n) CDR-H1 as set forth in SEQ ID NO: 203, CDR-H2 as set forth in SEQ ID NO: 204, CDR-H3 as set forth in SEQ ID NO: 205, CDR-L1 as set forth in SEQ ID NO: 206, CDR-L2 as set forth in SEQ ID NO: 207, and CDR-L3 as set forth in SEQ ID NO: 208; (o) CDR-H1 as set forth in SEQ ID NO: 214, CDR-H2 as set forth in SEQ ID NO: 215, CDR-H3 as set forth in SEQ ID NO: 216, CDR-L1 as set forth in SEQ ID NO: 217, CDR-L2 as set forth in SEQ ID NO: 218, and CDR-L3 as set forth in SEQ ID NO: 219; (p) CDR-H1 as set forth in SEQ ID NO: 226, CDR-H2 as set forth in SEQ ID NO: 227, CDR-H3 as set forth in SEQ ID NO: 228, CDR-L1 as set forth in SEQ ID NO: 229, CDR-L2 as set forth in SEQ ID NO: 230, and CDR-L3 as set forth in SEQ ID NO: 231; and (q) CDR-H1 as set forth in SEQ ID NO: 238, CDR-H2 as set forth in SEQ ID NO: 239, CDR-H3 as set forth in SEQ ID NO: 240, CDR-L1 as set forth in SEQ ID NO: 241, CDR-L2 as set forth in SEQ ID NO: 242, and CDR-L3 as set forth in SEQ ID NO:

243.

20. The process of claim 19, wherein the VH region comprises CDR-H1, CDR-H2, and CDR-H3, and the VL region comprises CDR-L1, CDR-L2, and CDR-L3 selected from the group consisting of:

25. 2. The process of claim 1, wherein the perfusion culture is carried out continuously for at least 7 days, preferably at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days, and most preferably at least 35 days, by supplying a defined cell specific perfusion rate and withdrawing excess cells from the bioreactor to maintain the biomass set point.

26. 10. An apparatus for performing a continuous upstream manufacturing process as described in claim 1, comprising a perfusion bioreactor, the first control loop, the second control loop, and an integrated unit.

27. 2. A bispecific antibody product produced by the upstream manufacturing process of claim 1.

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