Freeze-drying process by controlled ice nucleation for bispecific molecules.

Description

[0001] The disclosure relates to a method of freeze-drying a composition comprising bispecific molecules and the resulting freeze-dried composition. BACKGROUND

[0002] Bispecific molecules that engage T cells, such as BiTE® molecules, have demonstrated clinical benefits in immuno-oncology. Typically, one binding domain of these molecules is specific for a selected tumor-associated surface antigen on target cells, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. Due to their particular design, BiTE® molecules are especially suitable for transiently binding T cells to target cells while simultaneously potently activating the inherent cytolytic potential of T cells against target cells. Prior to storage, bispecific molecules that engage T cells are typically lyophilized. Lyophilization is a widely used manufacturing process to increase the stability of pharmaceutical products by removing the water content from a liquid product through lyophilization.Lyophilization is a batch process and is typically time-consuming, contributing more than 50% to the drug processing time. Furthermore, freeze dryers at manufacturing scale are limited in capacity; only a certain number of vials can be loaded in a batch, and there are limits to the amount of liquid composition that can be dispensed into the vials. Petition 870250078518, dated 03 / 09 / 2025, page 20 / 222 2 / 188 Due to these restrictions, lyophilized products are often expensive. See, for example, Awotwe-Otoo et al., International Journal of Pharmaceutics, 450 (2013), 70–78; Esfandiary et al., J. Pharm. Sci., 105 (2016), 1427–1433. Efforts to increase the efficiency of lyophilization processes for biopharmaceutical products, such as molecules that engage bispecific T cells, can be hampered by inconsistent product attributes, for example, excessive formation of high molecular weight species (HMWS), especially at higher product concentrations, resulting in costly product losses.

[0003] The freezing step in a conventional freeze-drying process involves uncontrolled or stochastic ice nucleation that occurs at a significantly lower temperature than the normal freezing point. Therefore, this supercooling results in small ice crystals, requiring long drying times. One approach to reduce the overall time of the freeze-drying process is to add an annealing step to the freeze-drying process, which increases the average size of the ice crystals through a process known as Ostwald ripening. However, as the present inventors have discovered, such an annealing step can lead to aggregation, i.e., the formation of high molecular weight species (HMWS), typically in high concentration bispecific molecule formulations, for example, at concentrations of at least 5 mg / ml or even at least 10 mg / ml, probably due to low-temperature degradation mechanisms.Therefore, there is a need for an improved freeze-drying process that provides a molecular product. Petition 870250078518, dated 03 / 09 / 2025, page 21 / 222 3 / 188 of bispecific T cell engagement with low aggregate product rates, even starting from higher concentration formulations. SUMMARY

[0004] In view of the unmet need described above, an object of the present invention is to provide a resource-saving method for delivering bispecific molecules, preferably bispecific molecules (involving T cells) comprising an Fc domain that prolongs the half-life, at concentrations above 10 mg / ml that exhibit only low levels (e.g., less than or equal to 1.5% of the total molecule) of undesirable aggregates, such as HMWS. The problem is solved by introducing a controlled ice nucleation (CIN) step into a freeze-drying process to freeze-dry high-concentration bispecific molecules with an Fc domain, to reduce the overall process time compared to standard freeze-drying processes. Said reduction in process time decreases energy consumption and frees up the apparatus earlier for the next run.In a first aspect, it is foreseen in the context of the present invention to provide a method for preparing a lyophilized bispecific molecule composition, the method comprising (a) a step (CIN) of inducing ice nucleation in a liquid bispecific molecule composition having a bispecific molecule concentration of at least 10 mg / ml in a vial exposed to a first temperature of about -10 °C to about -18 °C for about 60 minutes to about 270 minutes, wherein this step preferably comprises. Petition 870250078518, dated 03 / 09 / 2025, p. 22 / 222 4 / 188 a post-nucleation retention of up to 90 minutes, preferably about 30 minutes, (b) a freezing step of exposing the vial to a second temperature of about -25 °C to -50 °C for about 1 to 5 hours; (c) a drying step of composition (b) at a third temperature of about -5 °C to about -25 °C for about 25 to 70 hours; and (d) further drying composition (c) at a fourth temperature of about 25 °C to 50 °C for about 4 to 12 hours, resulting in a vial comprising a lyophilized bispecific molecule composition having a percentage content of high molecular weight species (HMWS) less than or equal to 1.5% (w / v), wherein the bispecific molecule comprises at least three domains, in which: A first domain binds to a tumor antigen on a target cell; A second domain binds to an extracellular epitope of the human and / or Macaca CD3e chain; and a third domain, which preferentially provides an extended half-life of the bispecific molecule, is fused to the second domain by a peptide linker, wherein the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain, wherein said two polypeptide monomers are fused to each other by means of a peptide linker.

[0005] Within the said aspect, it is also provided in the context of the present invention to provide a method, in which step (a) comprises exposing the flask to the first temperature for about 90 minutes to about three hours (step CIN). Petition 870250078518, dated 03 / 09 / 2025, page 23 / 222 5 / 188

[0006] Within the said aspect, it is also provided in the context of the present invention to provide a method, in which step (a) comprises exposing the flask to the first temperature for about 90 minutes to about two hours, preferably 110 minutes.

[0007] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which the first temperature in step (a) is from about -12 °C to about -17 °C.

[0008] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which the first temperature in step (a) is about -15 °C.

[0009] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein step (a) further comprises maintaining the bispecific molecule composition nucleated in ice at temperature for a post-nucleation time period of up to 90 minutes.

[0010] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method in which the post-nucleation time period is from about 20 minutes to about 90 minutes.

[0011] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method in which the post-nucleation time period is approximately 30 minutes.

[0012] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method wherein step (b) comprises exposing the flask to a second temperature of about -45 °C (freezing step).

[0013] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method, in which the Petition 870250078518, dated 03 / 09 / 2025, p. 24 / 222 6 / 188 The second time period is approximately two hours to approximately four hours.

[0014] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method of claim 10, in which the second time period is about three hours.

[0015] Within the said aspect, it is also provided in the context of the present invention to provide a method in which the first temperature is transitioned to the second temperature at a rate of about 0.01 °C to about 0.5 °C per minute.

[0016] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which the first temperature is transitioned to the second temperature at a rate of about 0.2 °C per minute.

[0017] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method, in which the third temperature of step (c) is from about 0 °C to about -20 °C (drying step), preferably from about 70 to 120 mTorr, preferably 100 mTorr.

[0018] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which the third temperature of step (c) is from about -5 °C to about -10 °C.

[0019] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which the third temperature of step (c) is about -8 °C.

[0020] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method, wherein step (c) comprises exposing the bottle comprising the Petition 870250078518, dated 03 / 09 / 2025, p. 25 / 222 7 / 188 composition of bispecific molecule at a temperature increase at a rate of about 0.01 °C to about 0.5 °C per minute.

[0021] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein the transition from step (b) to step (c) comprises increasing the temperature at a rate of about 0.2 °C to about 0.7 °C per minute and maintaining the flask at a temperature of about -40 °C to about -30 °C for about 15 minutes to about one hour.

[0022] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein the liquid composition of the bispecific molecule is exposed in step (a) to a first temperature of about -15 °C for about 110 minutes plus about 30 minutes after nucleation, is exposed in step (b) to a second temperature of about -45 °C for about 3 hours; the composition of (b) is dried in step (c) at a third temperature of about -8 °C for about 50 hours; and the composition of (c) is further dried at a fourth temperature of about 40 °C for about 8 hours, wherein step (d) is preferably carried out at about 70 to 120 mTorr, preferably 100 mTorr.

[0023] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method in which the bispecific molecule is a single-chain molecule.

[0024] Within the said aspect, it is also foreseen in the context of the present invention to provide a method in which the bispecific molecule is present in the composition at a concentration of about 10 mg / ml - 30 mg / ml, or about 20 Petition 870250078518, dated 03 / 09 / 2025, p. 26 / 222 8 / 188 mg / ml to approximately 30 mg / ml or, preferably, approximately 15 mg / ml to 25 mg / ml.

[0025] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein the method results in a vial comprising a lyophilized bispecific molecule composition exhibiting aggregation in terms of formation of high molecular weight species (HMSW) less than or equal to about 1.5% (w / w) or preferably less than or equal to about 1.2, 1, 0.75 or even 0.5% (relative to the total weight of the bispecific molecule).

[0026] Within the said aspect, it is also provided in the context of the present invention to provide a method, in which the third domain comprises an amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0027] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein each of the polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 249-256 or identical to a sequence selected from the group consisting of: SEQ ID NO: 249-256.

[0028] Within the aforementioned aspect, it is also foreseen in the context of the present invention to provide a method in which the first domain binds to CD33, CDH19, MSLN, FLT3, BCMA, CD19, MUC17, CDH3, CLDN18.2, CD70, EGFRviii, EpCAM, DLL3 and / or PSMA, preferably DLL3.

[0029] Within the said aspect, it is also provided in the context of the present invention to provide a method, wherein the first binding domain of the construct comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 and a region Petition 870250078518, dated 03 / 09 / 2025, page 27 / 222 9 / 188 VL comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: (a) CDR-H1 conform represented by SEQ ID NO: 4, CDR-H2 conform represented by SEQ ID NO: 5, CDR-H3 conform represented by SEQ ID NO: 6, CDR-L1 conform represented by SEQ ID NO: 1, CDR-L2 conform represented by SEQ ID NO: 2 and CDR-L3 conform represented by SEQ ID NO: 3, (b); CDR-H1 conform represented by SEQ ID NO: 29, CDR-H2 conform represented by SEQ ID NO: 30, CDR-H3 conform represented by SEQ ID NO: 31, CDR-L1 conform represented by SEQ ID NO: 34, CDR-L2 conform represented by SEQ ID NO: 35 and CDR-L3 conform represented by SEQ ID NO: 36, (c); CDR-H1 conform represented by SEQ ID NO: 42, CDR-H2 conform represented by SEQ ID NO: 43, CDR-H3 conform represented by SEQ ID NO: 44, CDR-L1 conform represented by SEQ ID NO: 45, CDR-L2 conform represented by SEQ ID NO: 46 and CDR-L3 conform represented by SEQ ID NO: 47, (d); CDR-H1 conform represented by SEQ ID NO: 53, CDR-H2 conform represented by SEQ ID NO: 54, CDR-H3 conform represented by SEQ ID NO: 55,CDR-L1 as represented in SEQ ID NO: 56, CDR-L2 as represented in SEQ ID NO: 57 and CDR-L3 as represented in SEQ ID NO: 58, (e) CDR-H1 as represented in SEQ ID NO: 65, CDR-H2 as represented in SEQ ID NO: 66, CDR-H3 as represented in SEQ ID NO: 67, CDR-L1 as represented in SEQ ID NO: 68, CDR-L2 as represented in SEQ ID NO: 69 and CDR-L3 as represented in SEQ ID NO: 70, (f) CDR-H1 as represented in SEQ ID NO: 83, CDR-H2 as represented in SEQ ID NO: 84, CDR-H3 as represented in SEQ ID NO: 85, CDR-L1 as represented, Petition 870250078518, dated 03 / 09 / 2025, page 28 / 222 10 / 188 as depicted in SEQ ID NO: 86, CDR-L2 as depicted in SEQ ID NO: 87 and CDR-L3 as depicted in SEQ ID NO: 88, (g) CDR-H1 as depicted in SEQ ID NO: 94, CDR-H2 as depicted in SEQ ID NO: 95, CDR-H2 as depicted in SEQ ID NO: 95, CDR-H3 as depicted in SEQ ID NO: 96 CDR-L1 as depicted in SEQ ID NO: 97, CDR-L2 as depicted in SEQ ID NO: 98 and CDR-L3 as depicted in SEQ ID NO: 99, (h) CDR-H1 as depicted in SEQ ID NO: 105, CDR-H2 as depicted in SEQ ID NO: 106, CDR-H2 as depicted in SEQ ID NO: 106, CDR-H2 as depicted in SEQ ID NO: 106, CDR-H2 as depicted in SEQ ID NO: 106 107, CDR-L1 as depicted in SEQ ID NO: 109, CDR-L2 as depicted in SEQ ID NO: 110 and CDR-L3 as depicted in SEQ ID NO: 111, (i) CDR-H1 as depicted in SEQ ID NO: 115, CDR-H2 as depicted in SEQ ID NO: 13, CDR-H2 as depicted in SEQ ID NO: 13 SEQ ID NO: 117, CDR-L1 as depicted in SEQ ID NO: 118, CDR-L2 as depicted in SEQ ID NO: 119 and CDR-L3 as depicted in SEQ ID NO: 120,(j) CDR-H1 as represented in SEQ ID NO: 126, CDR-H2 as represented in SEQ ID NO: 127, CDR-H3 as represented in SEQ ID NO: 128, CDR-L1 as represented in SEQ ID NO: 129, CDR-L2 as represented in SEQ ID NO: 130 and CDR-L3 as represented in SEQ ID NO: 131, (k) CDR-H1 as represented in SEQ ID NO: 137, CDR-H2 as represented in SEQ ID NO: 138, CDR-H3 as represented in SEQ ID NO: 139, CDR-L1 as represented in SEQ ID NO: 140, CDR-L2 as represented in SEQ ID NO: 141 and CDR-L3 as represented in SEQ ID NO: 142, (l) CDR-H1 as represented in SEQ ID NO: 152, CDR-H2 as represented in SEQ ID NO: 153, CDR-H3 as represented in SEQ ID NO: 154, CDR-L1 as represented, Petition 870250078518, dated 03 / 09 / 2025, page 29 / 222 11 / 188 as depicted in SEQ ID NO: 155, CDR-L2 as depicted in SEQ ID NO: 156 and CDR-L3 as depicted in SEQ ID NO: 157, (m) CDR-H1 as depicted in SEQ ID NO: 167, CDR-H2 as depicted in SEQ ID NO: 168, CDR-H2 as depicted in SEQ ID NO: 168, CDR-H2 as depicted in SEQ ID NO: 168, as depicted in SEQ ID NO: 163 169, CDR-L1 as depicted in SEQ ID NO: 170, CDR-L2 as depicted in SEQ ID NO: 171 and CDR-L3 as depicted in SEQ ID NO: 172, (n) CDR-H1 as depicted in SEQ ID NO: 203, CDR-H2 as depicted in SEQ ID NO: 204, CDR-H2 as depicted in SEQ ID NO: 204 SEQ ID NO: 205, CDR-L1 as depicted in SEQ ID NO: 206, CDR-L2 as depicted in SEQ ID NO: 207 and CDR-L3 as depicted in SEQ ID NO: 208; (o) CDR-H1 as depicted in SEQ ID NO: 214, CDR-H2 as depicted in SEQ ID NO: 215, CDR-H3 as depicted in SEQ ID NO: 216, CDR-L1 as depicted in SEQ ID NO: 217, CDR-L2 as depicted in SEQ ID NO: 218 and CDR-L2 as depicted in SEQ ID NO: 218 and CDR-L as depicted in SEQ ID NO: 213 NO: 219; (p) CDR-H1 as represented in SEQ ID NO: 226, CDR-H2 as represented in SEQ ID NO: 227, CDR-H3 as represented in SEQ ID NO: 228, CDR-L1 as represented in SEQ ID NO: 229, CDR-L2 as represented in SEQ ID NO: 230 and CDR-L3 as represented in SEQ ID NO: 231; and (q) CDR-H1 as represented in SEQ ID NO: 238, CDR-H2 as represented in SEQ ID NO: 239, CDR-H3 as represented in SEQ ID NO: 240, CDR-L1 as represented in SEQ ID NO: 241, CDR-L2 as represented in SEQ ID NO: 242 and CDR-L3 as represented in SEQ ID NO: 243.

[0030] Within said aspect, it is also provided in the context of the present invention to provide a method, wherein the first domain has an amino acid sequence Petition 870250078518, dated 03 / 09 / 2025, page 30 / 222 12 / 188 selected from the group consisting of the following SEQ ID numbers: 7, 8, 17, 27, 28, 37, 38, 39, 40, 41, 48, 49, 50, 51, 52, 59, 60, 61, 62, 63, 64, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93, 100, 101, 102, 103, 104, 113, 114, 121, 122, 123, 124, 125, 131, 132, 133, 134, 135, 136, 143, 144, 145, 146, 147, 148, 149, 150, 151, 158, 159, 160, 161, 162, 163, 164, 165, 166, 173, 174, 175, 176, 177, 178, 179, 180, 181, 223, 235 and 246, preferably 100 to 104.

[0031] Within the aforementioned aspect, it is also provided in the context of the present invention to provide a method in which ice nucleation is induced by means of ice mist or depressurization.

[0032] In a further aspect of the present invention, a lyophilized bispecific molecule composition is prepared by means of the method described in this document.

[0033] It should be understood that, although various modalities in the descriptive report are presented using the language “comprising,” in various circumstances, a related modality may also be described using the language “consisting of” or “essentially consisting of.” The disclosure contemplates modalities described as “comprising a feature” as including modalities that “consist of” or “essentially consist of” the feature. The term “a” or “an” refers to one or more. As such, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably in this document. The term “or” should be understood as encompassing items in the alternative form or jointly, unless the context unequivocally requires otherwise. Petition 870250078518, dated 03 / 09 / 2025, p. 31 / 222 13 / 188

[0034] It should also be understood that when describing a range of values, the disclosure contemplates individual values ​​found within the range. For example, “a pH of about pH 4 to about pH 6 may be, but is not limited to, pH 4.2, 4.6, 5.2, 5.5, etc. and any value between these values. In any of the ranges described in this document, the endpoints of the range are included in the range. However, the description also contemplates the same ranges in which the lower and / or upper endpoints are excluded. When the term “about” is used, it means the number mentioned plus or minus 5%, 10% or more of that number mentioned. The actual intended variation is determinable from the context.

[0035] Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of the present application, including the figures and detailed description, and all such features are considered aspects of the invention. Similarly, the features of the invention described herein may be recombined into additional embodiments which are also considered aspects of the invention, regardless of whether the combination of features is specified as an aspect or embodiment of the invention. The entire document is intended to be related as a unified disclosure and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combination of features is not found together in the same sentence, paragraph, or section of the present document. Furthermore, only such limitations as are described herein Petition 870250078518, dated 03 / 09 / 2025, p. 32 / 222 14 / 188 document as criticisms of the invention should be viewed as such; variations of the invention without limitations that have not been described in this document as criticisms are considered aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A-B: (A) A standard freeze-drying cycle for bispecific molecules without a CIN step contains annealing during the freeze-drying phase and a longer primary drying time. (B) Without an annealing step, CIN freeze-drying cycles rely on the induction of ice nucleation at higher temperatures to form larger ice crystals, resulting in shorter primary drying and total cycle times.

[0037] Figure 2A-C: Freeze-drying profiles of freeze-drying cycles for samples of bispecific molecules BCMAxCD3 and DLL3xCD3. The same freeze-drying tray arrangement was used for each freeze-drying cycle. (A) Standard cycle without CIN with annealing, where the maximum product temperature (Tp) of the bispecific molecule BCMAxCD3 was -33.3 °C and the maximum Tp of the bispecific molecule DLL3xCD3 was -32.8 °C. (B) Standard cycle without CIN and without annealing, in which the maximum product temperature (Tp) of the bispecific molecule BCMAxCD3 was -31.3 °C and the maximum Tp of the bispecific molecule DLL3xCD3 was -31.5 °C. (C) CIN cycle, where the maximum product temperature (Tp) of the bispecific molecule BCMAxCD3 was -27.8 °C and the maximum Tp of the bispecific molecule DLL3xCD3 was -28.7 °C.

[0038] Figure 3A-B: Change in % of HMWS after lyophilization for the standard cycle without CIN, the standard cycle without CIN and without annealing, and two CIN cycles 1 and 2 with different Petition 870250078518, dated 03 / 09 / 2025, p. 33 / 222 15 / 188 nucleation conditions and post-nucleation retentions. Due to this, the nucleation parameters of the CIN 2 cycle of -15 °C and 0.5 h post-nucleation were chosen to proceed with the experiments. (A) Bispecific molecule BCMAxCD3 (B) Bispecific molecule DLL3xCD3.

[0039] Figure 4A-B: Stability results of % HMWS over time for the standard cycle without CIN, the standard cycle without CIN and without annealing, and the CIN cycle for the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3. Values ​​are % HMWS recorded immediately after recognition at each time point. The lines are superimposed on the graph. (A) Bispecific molecule BCMAxCD3 (B) Bispecific molecule DLL3xCD3.

[0040] Figure 5: Moisture content at time zero of the bispecific molecule samples BCMAxCD3 and DLL3xCD3 after lyophilization. There was no significant difference in moisture content between the groups (p > 0.05).

[0041] Figure 6A-F: CEX-HPLC results of the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3. (A) Main peak of the bispecific molecule BCMAxCD3 (B) Acid peak of the bispecific molecule BCMAxCD3 (C) Basic peak of the bispecific molecule BCMAxCD3 (D) Main peak of the bispecific molecule DLL3xCD3 (E) Acid peak of the bispecific molecule DLL3xCD3 (F) Basic peak of the bispecific molecule DLL3xCD3

[0042] Figure 7: Average cake resistance at 1 mm depth in a 6R vial with a filling volume of 1.3 ml. Cake resistance is >50% lower when using CIN compared to the standard freeze-drying process and even significantly reduced compared to the process Petition 870250078518, dated 03 / 09 / 2025, page 34 / 222 16 / 188 standard with additional annealing. The “CIN 1” process with CIN step parameters not according to the present invention had a nucleation temperature of -7 °C and, in contrast, the “CIN 2” process according to the present invention had a nucleation temperature of -15 °C. DETAILED DESCRIPTION

[0043] The disclosure provides a method for preparing a lyophilized bispecific molecule composition, in particular a composition comprising a bispecific T cell engagement molecule comprising a half-life extending Fc domain, the method comprising a controlled ice nucleation (CIN) step. Surprisingly, CIN was found to be a better alternative to introducing an annealing step in the lyophilization of bispecific molecules according to the present invention in order to achieve both the desired product quality (i.e., significant reduction in HMWS compared to conventional lyophilization with an annealing step) and reduced process time compared to a conventional process without an annealing step or CIN step. The process time savings come from a shorter step (c) and (d), as described herein, compared to a conventional lyophilization process without annealing.For example, a conventional freeze-drying process can take up to 100 hours for the primary drying step (c), while a process according to the present invention has a shorter drying time, preferably only about 50 hours for step (c). Considering the secondary drying step (d), the overall time savings of a process is... Petition 870250078518, dated 03 / 09 / 2025, page 35 / 222 17 / 188 of at least 30% or even more, such as 40%, compared to a conventional freeze-drying process. Therefore, resource consumption such as energy is also reduced. An exemplary conventional freeze-drying process or – also referred to in this document as a standard freeze-drying process, i.e., generally without a CIN step, for bispecific molecules, is understood in this document as not having a CIN or annealing step. a freezing step (b) of, for example, about -45 °C for about 2 hours, a primary drying step (c), for example, at about -25 °C and about 70 mTorr for about 100 hours and a secondary drying step (d), for example, at about 40 °C and about 70 mTorr for about 8 hours.

[0044] Without wanting to get bogged down in theory, larger ice crystals and therefore shorter cycle times are obtained by introducing the CIN step with the process parameters as revealed in this document.CIN introduces externally formed ice crystals into the drug vials, resulting in larger ice crystals after freezing. In the present invention, it is demonstrated that the application of a CIN step, in contrast to, for example, an annealing step operated under selected conditions targeting bispecific molecules, efficiently attenuates aggregation in terms of HMWS, without impacting other product quality attributes such as lyophilization cake appearance and moisture content. Furthermore, resistance to agglomeration was advantageously reduced, for example, by at least 30%, typically by about 50% by the present method comprising a CIN step compared to standard lyophilization without CIN with or without a step of. Petition 870250078518, dated 03 / 09 / 2025, page 36 / 222 18 / 188 Annealing. The freeze-drying cycle with annealing normally causes undesirable aggregation, typically at least 3% HMSW, whereas a freeze-drying process comprising a CIN step, as described in this document, keeps the aggregation, i.e., HMSW, at a level equal to or less than 1.5%, which is close to the pre-freeze-drying value of 1% or even lower, as demonstrated in the examples.

[0045] The CIN step according to the present invention comprises incubation preferably in the range of -18 to -10 °C for about 1 to 5 hours, typically 90 min to 120 min, such as 90, 110 or 120 min, to cool the composition (pre-nucleation) so that the ice crystals do not melt when introduced in the next step. Ice crystals are introduced to induce ice nucleation by incubation, preferably in the range of -18 to -10 °C for a longer time to allow the ice crystals to grow (post-nucleation). It is an advantage of the present invention to provide a method comprising a CIN step based on “sweet spot” conditions that facilitate both the beneficial quality of the bispecific molecular product (in terms of product uniformity, preferably without HMWS, at higher product concentrations) as explained and demonstrated herein, and make the process more efficient in relation to the shorter drying process duration.Higher ice nucleation temperatures above -10 °C, such as -7 °C, despite the advantage of faster drying time, generally result in bispecific molecular products with lower product quality in terms of aggregation. In contrast, a lower ice nucleation temperature... Petition 870250078518, dated 03 / 09 / 2025, page 37 / 222 19 / 188 °C can typically lead to longer drying times and may negate any benefits in terms of process duration and resources, as well as energy savings. Some process parameters may have less impact on product quality and process economics in terms of drying time than the temperature and duration of the CIN step. For example, the secondary drying pressure cannot be set over a wider range, as it is known in the art that secondary drying pressure has less impact on the drying process and therefore may not serve to save energy.

[0046] The method preferably comprises inducing ice nucleation in a liquid bispecific molecule composition in a flask exposed to a first temperature in the range of about -18°C to about -10°C (e.g., about -18, -17, -16, -15, -14, -13, -12, -11 or -10 °C) for about 1 to about 5 hours, preferably 90 to 120 minutes; exposing the flask to a second temperature of about -25°C to -50°C for a second period of time; and drying the composition at a third temperature of about -5°C to about -25°C. The method results in a vial comprising a lyophilized bispecific molecule composition with advantageous product parameters (low percentage of HMWS despite higher product concentration and commercially beneficial) and having an aspect ratio greater than or equal to about 0.75 (e.g., greater than or equal to 0.8, 0.85, 0.9, or 0.95).In several respects, the method results in a vial comprising a lyophilized bispecific molecule composition with an aspect ratio greater than or equal to about 1 (e.g., greater than or equal to). Petition 870250078518, dated 03 / 09 / 2025, p. 38 / 222 20 / 188 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2). Notably, the process disclosed in this document allows for a surprisingly high fill volume for a given vial (i.e., aspect ratios), while minimizing the risks of vial breakage and maintaining reasonable drying times. “Aspect ratio is the fill height of a vial (the height of the bispecific molecular composition in the vial) divided by the inner diameter of the vial (aspect ratio = fill height / inner diameter of the vial). Previous methods required introducing reduced volumes into the vials (a lower aspect ratio) to avoid vial breakage and product cake collapse during the freeze-drying process. The method described in this document allows for higher aspect ratios during the freeze-drying process, which provides a number of potential advantages.For example, for bispecific molecule therapies administered in large doses, fewer dosing vials are needed. Alternatively (or additionally), the process allows the use of smaller vials compared to other lyophilization methods, while maintaining the same filling volume, resulting in more units per batch and consequently higher production. The use of smaller vials also offers the benefit of reducing storage space in manufacturing facilities and clinics. These are just examples of the advantages of the flash method, which are obtained during the production of boluses of products suitable for biopharmaceutical use.

[0047] The disclosure describes various conditions for use in a freeze-drying process to produce a freeze-dried bispecific molecule composition. Generally, vials (by Petition 870250078518, dated 03 / 09 / 2025, page 39 / 222 21 / 188 example, glass vials) suitable for pharmaceutical compositions are filled with a liquid bispecific molecular composition and exposed to different temperatures and pressures to obtain a lyophilized product. The vial can be of any size or shape suitable for use in lyophilization processes and can be formed from a variety of materials such as glass, metal, or plastic (e.g., polycarbonate, polystyrene, polypropylene, or polyolefin). For example, the vial may be glass or glass-like and have a tubular shape. Molded glass vials are commercially available in a variety of different sizes and dimensions. In fact, several vial sizes are commercially available (e.g., size 2R, 4R, 6R, 8R, 10R, 15R, 20R, 25R, 30R, 50R, or 100R). The bottle can be constructed to include a suitable stopper, such as a commercially available elastomeric stopper, for example, from Daikyo Seiko, Ltd.or at West Pharmaceutical Services, Inc. The steps described in this document are conducted, in many respects, in a freeze-drying chamber or ice nucleation system. Freeze-drying chambers may be operated according to the manufacturer's instructions appropriate for pharmaceutical compositions. In several aspects of the disclosure, the freeze-drying method does not comprise an annealing step. Annealing refers to a process in which the temperature of the formulation is changed (e.g., from a low temperature to a higher temperature, and then back to a low temperature). Several aspects of the disclosed method allow for the production of a freeze-dried product without this annealing step. Petition 870250078518, dated 03 / 09 / 2025, page 40 / 222 22 / 188

[0048] The development method comprises inducing ice nucleation in a liquid bispecific molecule composition in a flask (referred to herein as the “ice nucleation step”). Ice nucleation can be initiated using any of several methods, including, but not limited to, ice fog, sudden / rapid depressurization, and vacuum-induced evaporative cooling. Other controlled ice nucleation methods include, for example, ultrasound, interval freezing, electrical freezing, temperature quenching freezing, use of a pre-chilled shelf, and mechanical agitation.

[0049] In several aspects of the development, ice nucleation is induced in the liquid bispecific molecule composition by means of ice mist. Ice misting involves seeding a supercooled solution in vials with externally generated ice crystals. An ice mist generator is used to produce a fine suspension of ice crystals that is injected into the freeze dryer chamber. The ice mist crystals serve as ice seeds for the supercooled liquid product in the vials. As soon as the ice crystals from the mist enter the partially capped vials and come into contact with the surface of the supercooled liquid, ice nucleation occurs instantaneously within the vial at a specified shelf temperature. This occurs simultaneously in all vials, improving intra-batch homogeneity. Ice mist systems are available from IMA Life (Tonawanda, NY) and Millrock Technologies (Kingston, NY). The technology Petition 870250078518, dated 03 / 09 / 2025, page 41 / 222 23 / 188 of ice fog is further described in, for example, Azzarella et al., BioPharm. Int., 29(12) (2017), 36-41.

[0050] In several aspects of the revelation, ice nucleation is induced in the liquid bispecific molecule composition by means of depressurization. Rapid depressurization generally involves first pressurizing a lyophilizer chamber to 1.5 to 2 atmospheres (about 20-30 psig) using an inert gas such as nitrogen, and then rapidly releasing (e.g., in 3 seconds or less) the pressure to slightly above ambient. The rapid change in pressure induces nucleation in the vials. Rapid depressurization systems are available from SP Scientific (Gardiner, NY) and described in more detail in, for example, Luoma et al., Controlled Ice Nucleation Using ControLyo® Pressurization-Depressurization Method, In: Ward K., Matejtschuk P. (eds) Lyophilization of Pharmaceuticals and Biologicals. Methods in Pharmacology and Toxicology. Humana Press, New York, NY, 2019, pp. 57-77.

[0051] Vacuum-induced evaporative cooling generally involves reducing the pressure of the freeze-drying chamber to slightly above the boiling point of the solution, allowing the enhanced evaporative cooling effect of the liquid surface to cause nucleation. Vacuum-induced evaporative cooling systems are available from HOF Sonderanlagenbau GmbH (Lohra, Germany).

[0052] Ice nucleation (e.g., via ice fog) is induced in the liquid bispecific molecular composition in the context of the present invention in a flask exposed to an initial temperature of about -18°C to about -10°C. Petition 870250078518, dated 03 / 09 / 2025, page 42 / 222 24 / 188°C (such as approximately -18°C, approximately -17°C, approximately -16°C, approximately -15°C, approximately -14°C, approximately -13°C, approximately -12°C, approximately -11°C, or approximately -10°C) for a period of time of approximately 60 minutes to approximately five hours. Optionally, the ice nucleation step comprises exposing the vial to the first temperature for approximately 30 minutes to approximately two hours, for example, approximately 90 minutes to approximately two hours. In many respects, the vial is exposed to a first temperature of approximately -15°C. A higher temperature, such as -7°C, leads to significantly higher percentage levels of HMWS, which is undesirable in the context of the present invention nor in the production of bispecific therapeutic molecules in general. Therefore, the careful selection of specific temperature ranges for the CIN step is not arbitrary.Optionally, this step of the method further comprises maintaining the composition of the bispecific molecule nucleated on ice at the temperature for a post-nucleation period of up to two hours (optionally at the same temperature). For example, the post-nucleation holding time can be from about 30 minutes to about 90 minutes (e.g., about 45 minutes to about 75 minutes, such as 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, or 75 minutes). In several respects, the post-nucleation holding time is about 60 minutes.

[0053] The method further comprises exposing the flask to a second temperature of about -25°C to about -50°C for a second period of time (referred to herein as a “freezing step”). The second temperature may be, for example, about -25°C to about -45°C, about -25°C to about -40°C, or about -25°C. Petition 870250078518, dated 03 / 09 / 2025, p. 43 / 222 25 / 188 at approximately -35°C, approximately -35°C to approximately -50°C, approximately -40°C to approximately -50°C, or approximately -45°C to approximately 50°C (such as approximately -40°C, approximately -41°C, approximately -42°C, approximately -43°C, approximately -44°C, approximately -45°C, approximately -46°C, approximately -47°C, approximately -48°C, approximately -49°C, or approximately -50°C). In several respects, the freezing step involves exposing the flask to a second temperature of approximately -45°C. The second time period is optionally from approximately one hour to approximately five hours, as well as from approximately two hours to approximately four hours. The second time period may be approximately 60 minutes, approximately 90 minutes, approximately 120 minutes, approximately 150 minutes, approximately 180 minutes, approximately 210 minutes, or approximately 240 minutes (or any interval comprising these endpoints). In many respects, the second time period is approximately three hours.

[0054] The cooling rate of the vials can be controlled in several aspects of the development. For example, the first temperature is optionally transferred to the second temperature at a rate of about 0.01°C to about 0.5°C per minute (e.g., about 0.05°C to about 0.45°C per minute, about 0.1°C to about 0.3°C per minute, or about 0.15°C to about 0.25°C per minute). In several aspects, the first temperature is transferred to the second temperature at a rate of about 0.2°C per minute.

[0055] The method further comprises drying the composition resulting from the freezing step at a third temperature of about 0°C to about 40°C (referred to in this document as a “drying step”), resulting Petition 870250078518, dated 03 / 09 / 2025, p. 44 / 222 26 / 188 in a flask comprising a lyophilized bispecific molecule composition with an aspect ratio greater than or equal to about 0.75. In several respects, the third temperature used in the drying step is about 0°C to about 35°C, about 0°C to about 30°C, about 5°C to about 40°C, about 10°C to about 40°C, about 15°C to about 40°C, about 20°C to about 40°C, about 25°C to about 40°C, about 30°C to about 40°C or about 35°C to about 40°C. Optionally, the third temperature ranges from approximately 0°C to approximately 25°C, such as approximately 10°C to approximately 25°C (for example, approximately 25°C).

[0056] Optionally, the transition from the freezing stage to the drying stage comprises increasing the temperature at a rate of about 0.2°C to about 0.7°C per minute and maintaining the flask at a temperature of about -40°C to about -30°C for about 15 minutes to about one hour. For example, the transition may comprise increasing the temperature at a rate of about 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C or 0.7°C. The transition may occur over a period of time of, for example, about 15 minutes, 30 minutes, 45 minutes or 60 minutes.

[0057] The drying step may, in various aspects of the development, comprise exposing the vial comprising the bispecific molecular composition to a temperature increase at a rate of about 0.01°C to about 0.5°C per minute (for example, about 0.05°C to about 0.45°C per minute, about 0.1°C to about 0.3°C per minute or about 0.15°C to about 0.25°C per minute). In various aspects of the development, the drying step comprises (1) Petition 870250078518, dated 03 / 09 / 2025, p. 45 / 222 27 / 188 maintain the vial at a temperature of about -5°C to about 5°C for about 8 hours to about 12 hours and (2) maintain the vial at a temperature of about 20°C to about 30°C for about 20 hours to about 50 hours. In exemplary aspects of the development, drying step 1 comprises maintaining the vial at a temperature of about -5°C to about 0°C, about 0°C to about 5°C or about -2°C to about 2°C; such as about -5°C, about -4°C, about -3°C, about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C or any range with these endpoints. The time period for drying stage 1 is, in various respects, from about 8 hours to about 10 hours, from about 9 hours to about 11 hours, or from about 10 hours to about 12 hours; such as about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours, or any interval with these endpoints.In exemplary aspects of the development, drying step 2 comprises maintaining the vial at a temperature of about 25°C to about 30°C, about 20°C to about 25°C or about 23°C to about 27°C; such as about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C or about 30°C or any interval with these endpoints. The time period for drying step 2 is optionally from approximately 20 hours to approximately 45 hours, approximately 20 hours to approximately 40 hours, approximately 20 hours to approximately 35 hours, approximately 20 hours to approximately 30 hours, approximately 25 hours to approximately 50 hours, approximately 30 hours to approximately 50 hours, approximately 35 hours to approximately 50 hours. Petition 870250078518, dated 03 / 09 / 2025, page 46 / 222 28 / 188 approximately 40 hours to approximately 50 hours or approximately 45 hours to approximately 50 hours (e.g., approximately 20 hours, approximately 25 hours, approximately 30 hours, approximately 35 hours, approximately 40 hours, approximately 45 hours or approximately 50 hours). The drying step (1) optionally comprises maintaining the vial at a temperature of approximately 0°C for approximately 10 hours. The drying step (2) optionally comprises maintaining the vial at a temperature of approximately 25°C for approximately 40 hours.

[0058] The development method allows the use of larger volumes of liquid composition in the vials before lyophilization. In this sense, the liquid composition of the bispecific molecule can fill at least 50% of the vial volume before performing the ice nucleation step. The method may involve, prior to the ice nucleation step, filling at least 50% of the flask volume with the liquid composition of bispecific molecules.The liquid composition of the bispecific molecule can fill at least 55%, 60%, 65%, or 75% of the vial.

[0059] The disclosure further provides a lyophilized bispecific molecule composition prepared using the method described in this document. The disclosure further provides a lyophilized bispecific molecule composition prepared using the method described in this document. The formulation also optionally has a pH of about 3 to about 7 (or about 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7). In some cases, the pH is about 4 to about 6. In some preferred cases, the pH of the formulation is about 4 or about 4.2. In several cases, the pH of the formulation is about 5. In some embodiments, the pH of the formulation is about 6. Petition 870250078518, dated 03 / 09 / 2025, p. 47 / 222 29 / 188

[0060] In some embodiments, the bispecific molecule of the lyophilized formulation is an antigen-binding protein. An antigen-binding protein is a protein comprising a domain that binds to a specified target antigen (such as HER2 or CD3 and / or DLL3, BCMA, or CD33). An antigen-binding protein comprises a template portion or structure that allows the antigen-binding domain to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen.

[0061] The term half-life is understood in this document as the time required for the concentration of a biological substance (such as the bispecific molecule of the invention) to decrease from its maximum concentration (Cmax) to half of Cmax in blood plasma. An extended half-life is preferably at least 40 hours, more preferably 50, 60, 70, 80, 90 or 100 hours compared to a bispecific molecule without an extended half-life which typically has a half-life of 24 hours or less.

[0062] The term polypeptide is understood in this document as an organic polymer comprising at least one continuous, unbranched amino acid chain. In the context of the present invention, a polypeptide comprising more than one amino acid chain is similarly provided. An amino acid chain of a polypeptide typically comprises at least 50 amino acids, preferably at least 100, 200, 300, 400 or 500 amino acids. It is also provided in the context of this invention that an amino acid chain of a polymer is linked to an entity that is not composed of amino acids. Petition 870250078518, dated 03 / 09 / 2025, p. 48 / 222 30 / 188

[0063] The term “antigen-binding polypeptide” according to the present invention is preferably a polypeptide that binds immunospecifically to its target or antigen. It typically comprises the variable region of the heavy chain (VH) and / or the variable region of the light chain (VL) of an antibody or comprises domains derived therefrom. A polypeptide according to the invention comprises the minimum structural requirements of an antibody that allow immunospecific binding to the target. This minimum requirement may, for example, be defined by the presence of at least three CDRs of the light chain (i.e., CDR1, CDR2 and CDR3 of the VL region) and / or three CDRs of the heavy chain (i.e., CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs.An antigen-binding molecule of the present invention is preferably a T-cell coupling polypeptide that can be characterized by the presence of three or six CDRs in one or both of the binding domains, and the skilled person knows where (in what order) these CDRs are located within the binding domain. Preferably, an “antigen-binding molecule” is understood as an “antigen-binding polypeptide” in the context of the present invention. In an alternative embodiment, an antigen-binding polypeptide of the present invention may be an aptamer.

[0064] Alternatively, a molecule in the context of the present invention is an antigen-binding polypeptide that corresponds to an antibody construct, which typically refers to a molecule in which the structure and / or function is / are based on the structure and / or function of an antibody, for example, an immunoglobulin molecule of length Petition 870250078518, dated 03 / 09 / 2025, p. 49 / 222 31 / 188 total or whole. An antigen-binding molecule is consequently capable of binding to its specific target or antigen and / or is / are extracted from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or its fragment. Furthermore, the domain that binds to its binding partner according to the present invention is understood herein as a binding domain of an antigen-binding molecule according to the invention. Typically, a binding domain according to the present invention comprises the minimum structural requirements of an antibody that allows binding to the target. This minimum requirement may, for example, be defined 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 approach to defining the minimum structural requirements of an antibody is to define the antibody epitope within the specific target structure, respectively, from the protein domain of the target protein comprising the epitope region (epitope cluster) or by reference to a specific antibody competing with the defined antibody epitope. Antibodies on which the constructs according to the invention are based include, for example, monoclonal, recombinant, chimeric, deimmunized, humanized, and human antibodies.

[0065] In the context of the present invention, a polypeptide of the present invention binds to its respective target structure in a specific manner. Preferably, a polypeptide according to the present invention comprises a Petition 870250078518, dated 03 / 09 / 2025, page 50 / 222 32 / 188 paratope by binding domain that “binds specifically or immunospecifically to,” “recognizes (specifically or immunospecifically),” or “reacts (specifically or immunospecifically) with its respective target structure.” This means, according to this invention, that a polypeptide or its binding domain interacts or interacts (immuno-)specifically with a given epitope on the target molecule (antigen) and CD3, respectively. This interaction or association occurs more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of these parameters with an epitope on the specific target than with alternative substances (non-target molecules). Due to the sequence similarity between homologous proteins in different species, a binding domain that binds (immuno-)specifically to its target (such as a human target) may, however, cross-react with homologous target molecules from different species (such as non-human primates).The term “specific / immunospecific binding” can therefore include the binding of a binding domain to epitopes and / or structurally related epitopes in more than one species. The term “binds (immuno-)selectively” excludes binding to structurally related epitopes.

[0066] The binding domain of an antigen-binding molecule according to the invention may, for example, comprise the aforementioned groups of CDRs. Preferably, these CDRs are comprised within the structure of a variable region of the antibody light chain (VL) and a variable region of the antibody heavy chain (VH); however, it does not have to comprise both. The fragments Petition 870250078518, dated 03 / 09 / 2025, page 51 / 222 33 / 188 Fd, for example, have two VH regions and often retain some antigen-binding function from the intact antigen-binding domain. Additional examples of antibody fragment shapes, antibody variants, or binding domains include (1) a Fab fragment, a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; (3) an Fd fragment having the two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody; (5) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which has a VH domain; (6) an isolated complementarity determining region (CDR) and (7) a single-string Fv (scFv), the latter being preferred (e.g., derived from an scFv library).Examples of antigen-binding molecule embodiments according to the invention are, for example, described in patent documents 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.

[0067] Also within the definition of “binding domain” or “binding domain” are full-length antibody fragments such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or “r IgG” (“half antibody”). Antigen-binding molecules according to the invention may also comprise modified antibody fragments, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-lock, scFab, Fab2, Fab3, Petition 870250078518, dated 03 / 09 / 2025, page 52 / 222 34 / 188 diabodies, single-chain diabodies, tandem diabodies (Tandabs), 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 merely a variable domain, which may be VHH, VH, or VL, that binds specifically to an antigen or epitope independently of other V regions or domains. Typically, a binding domain of the present invention comprises a paratope that facilitates binding to its binding partner.

[0068] As used in this document, the terms single-chain Fv, single-chain antibodies, or scFv refer to single-chain polypeptide antibody fragments comprising the variable regions of both heavy and light chains, but lacking the constant regions. Generally, a single-chain antibody additionally comprises a polypeptide linker between the VH and VL domains that allows it to form the desired structure that would permit 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). Several methods of generating single-chain antibodies are known, including those described in US Patent Documents Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Houston et al. (1988) Proc. Natl. Sci.EUA 85:587 9-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041. In specific modalities, single-stranded antibodies. Petition 870250078518, dated 03 / 09 / 2025, p. 53 / 222 35 / 188 can also be bispecific, multispecific, human and / or humanized and / or synthetic.

[0069] In the context of the present invention, a paratope is understood to be an antigen-binding site, which is part of a polypeptide, as described herein, and which recognizes and binds to an antigen. A paratope is typically a small region of at least 5 amino acids. A paratope, as understood herein, typically comprises parts of antibody-derived heavy chain (VH) and light chain (VL) sequences. Each binding domain of a polypeptide according to the present invention is provided with a paratope comprising a set of 6 complementarity-determining regions (CDR loops), with three of each comprised within the antibody-derived VH and VL sequence, respectively.

[0070] Furthermore, the definition of the term antigen-binding molecule preferably includes polyvalent / multivalent constructs and thus bispecific molecules, where bispecific means that they bind specifically to two types of cells comprising distinct antigenic structures, i.e., target cell(s) and effector cell(s). As the antigen-binding molecules of the present invention are preferably multidirectional, they are typically also polyvalent / multivalent molecules, i.e., they bind specifically to more than two antigenic structures, preferably four distinct binding domains in the context of the present invention, which are two target-binding domains and two CD3-binding domains. The term antigen-binding molecule Petition 870250078518, dated 03 / 09 / 2025, page 54 / 222 36 / 188 multi-targeting bispecific antigen comprises the terms multi-targeting bispecific T cell coupling molecule and multi-targeting bispecific T cell coupling polypeptide (MBiTEP). A multi-targeting bispecific antigen-binding molecule is either a multi-targeting bispecific T cell coupling molecule or a multi-targeting bispecific T cell coupling polypeptide (MBiTEP). The term multi-targeting bispecific T cell coupling molecule is understood to encompass the term multi-targeting bispecific T cell coupling polypeptide.Furthermore, the definition of the term antigen-binding molecule includes molecules comprising only one polypeptide chain as well as molecules consisting of more than one polypeptide chain, which chains may be identical (homodimers, homotrimers, or homo-oligomers) or different (heterodimer, heterotrimer, or hetero-oligomer). Such molecules comprising more than one polypeptide chain, that is, typically two chains, have these chains typically attached to each other as heterodimers through charged-pair linkage, for example, within a heteroFc entity that serves as a spacer and half-life extension fraction between the two bispecific entities as described in this document.Examples of the antigen-binding molecules identified above, for example, antibody-based molecules and their variants or derivatives, are described, among others, in Harlow and Lane, Antibodies: a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999). Petition 870250078518, dated 03 / 09 / 2025, page 55 / 222 37 / 188 Kontermann and Dübel, Antibody Engineering, Springer, 2aed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0071] The term bispecific as used herein refers to an antigen-binding molecule that is at least bispecific, i.e., addresses two different cell types, i.e., target and effector cells, and comprises at least a first and third binding domain and a second and fourth binding domain, wherein at least two binding domains bind to two antigens or targets preferably selected from CD20, CD22, FLT3, MSLN, CDH3, CLL1 and EpCAM and the other two binding domains of the same molecule bind to another antigen (herein: CD3) on an effector cell, typically a T cell. Consequently, the antigen-binding molecules according to the invention comprise specificities for at least two different antigens or targets. For example, two domains do not preferentially bind to an extracellular epitope of CD3e from one or more of the species as described herein.

[0072] The term target cell surface antigen refers to an antigenic structure expressed by a cell and present on the cell surface such that it is accessible to an antigen-binding molecule as described herein. A preferred target cell surface antigen in the context of the present invention is a tumor-associated antigen (TAA). 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 Petition 870250078518, dated 03 / 09 / 2025, page 56 / 222 38 / 188 glycoprotein. It is preferably a tumor antigen. The term “bispecific antigen-binding molecule” of the invention also encompasses bispecific multidirectional antigen-binding molecules as well as tridirectional antigen-binding molecules, the latter including three binding domains or constructs having more than three (e.g., four, five) specificities.

[0073] Preferred in the context of the present invention is a molecule that has “multitargeting,” which is understood herein as “targeting at least two targets (e.g., TAAs) per molecule of the invention, typically per target cell.” In this respect, a multitargeting molecule, such as an antigen-binding molecule, is specific to two typically identical effector structures—on an effector cell such as CD3, more preferably CD3epsilon (CD3e, which is understood whenever reference is made to “CD3” in the present invention)—and at least two target cell surface antigens. Said specificity is conferred by the respective binding domains as defined herein.Typically, "multitargeting" refers to a molecule that is specific to at least two (preferably different) target cell surface antigens (e.g., TAAs) that confers preferential properties of a multitargeting antigen-binding molecule according to the present invention, namely mitigation of antigen loss and increased selectivity, i.e., selectivity to kill target cells that co-express the targets for which the molecule of the invention has binding domains and those target cells that are associated with a disease. Petition 870250078518, dated 03 / 09 / 2025, p. 57 / 222 39 / 188 In this way, the therapeutic window of the molecule of the invention is increased with respect to bispecific, monotargeted molecules, which typically leads to higher drug tolerability, as demonstrated in this document.

[0074] A T-cell coupling antigen-binding molecule, for example, a single-chain polypeptide, according to the present invention is preferably bispecific, which is understood herein as typically comprising a domain binding to at least one target antigen and another domain binding to CD3. Consequently, it does not occur naturally and is markedly different in its function from naturally occurring products. A polypeptide according to the invention is therefore an artificial hybrid polypeptide comprising at least two distinct binding domains with different specificities and is thus bispecific. Bispecific antigen-binding molecules can be produced by a variety of methods including hybridoma fusion or Fab fragment ligation. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0075] The at least four binding domains and the variable domains (VH / VL) of the antigen-binding molecule of the present invention typically comprise peptide linkers (spacer peptides). The term peptide linker comprises, according to the present invention, an amino acid sequence whereby the amino acid sequences of one domain (variable and / or binding) and another domain (variable and / or binding) of the molecule of Petition 870250078518, dated 03 / 09 / 2025, page 58 / 222 The 40 / 188 antigen-binding domains of the invention are linked together. The peptide linker between the first and second binding domains and the third and fourth domains, wherein the first and third domains are preferably capable of simultaneously binding to two targets, which are preferably different targets (e.g., TAA1 and TAA2) preferably in the same cell, are preferably flexible and of limited length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids. Peptide linkers can also be used to fuse the spacer to the other domains of the antigen-binding molecule of the invention. An essential technical feature of such a peptide linker is that it does not comprise any polymerization activity. Suitable peptide linkers include those described in U.S. Patents Nos. 4,751,180 and 4,935,233 or WO 88 / 09344.Peptide linkers can also be used to attach other domains or modules or regions (such as half-life extension domains) to the antigen-binding molecule of the invention. However, typically the linker between the first and second target-binding domains differs from the intra-linker linker that links the VH and VL within the target-binding domain. This difference is that the linker between the first and second binding domains has one more amino acid than the intra-linker linkers, for example, six and five amino acids respectively, such as SGGGGS versus GGGGS. This surprisingly confers both flexibility and stability to the shape of the antigen-binding molecule. Petition 870250078518, dated 03 / 09 / 2025, p. 59 / 222 41 / 188 specific as described in this document. The spacer (or synonymously spacer entity) between the two bispecific entities as described in this document is a specific embodiment of a linker because a spacer also functions as a linker because it contributes to linking the two bispecific entities to preferentially construct at least one continuous polypeptide chain comprising the four linking domains or parts thereof. However, additionally, the spacer functions as an entity that sterically separates the two bispecific entities.Consequently, a spacer in the context of the present invention is a specific embodiment of a connector that – together with two additional short and flexible connectors at each end – contributes to connecting the two connection domains (of two different bispecific entities) but first of all separates them in such a way that the two bispecific entities can act advantageously as described in this document, for example, showing a surprisingly high selectivity gap.

[0076] The antigen-binding molecules of the present invention are preferably “in vitro generated antigen-binding molecules”. This term refers to an antigen-binding molecule according to the above definition where all or part of the variable region (e.g., at least one CDR) is generated in a selection of non-immune cells, for example, an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen. Petition 870250078518, dated 03 / 09 / 2025, p. 60 / 222 42 / 188 preferably, sequences generated only by genomic rearrangement in an immune cell in an animal. A recombinant antibody is an antibody made through the use of recombinant DNA technology or genetic manipulation.

[0077] The term monoclonal antibody (mAb) or monoclonal antibody from which an antigen-binding molecule as used in this document is derived refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations and / or post-translational modifications (e.g., isomerizations, amidations) which may be present in minute quantities.Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations which 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 hybridoma culture, and consequently not contaminated by other immunoglobulins. The monoclonal modifier indicates the antibody's character as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0078] For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. By Petition 870250078518, dated 03 / 09 / 2025, page 61 / 222 43 / 188 For example, monoclonal antibodies to be used may be made by the hybridoma method first described by Koehler et al., Nature, 256: 495 (1975), or they may be made by recombinant DNA methods (see, for example, US Patent Document No. 4,816,567). Examples of other techniques for producing human monoclonal antibodies include the triome 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).

[0079] Hybridomas can then be analyzed using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis, for example, Biacore™, to identify one or more hybridomas that produce an antibody that specifically binds to a specified antigen. Any form of the relevant antigen can be used as the immunogen, for example, recombinant antigen, naturally occurring forms, any of its variants or fragments, as well as its antigenic peptide. Surface plasmon resonance as employed in the Biacore system can be used to increase the efficiency of phage antibodies that bind to an epitope of a target cell surface antigen (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0080] Another illustrative method of producing monoclonal antibodies includes screening protein expression libraries, for example, phage display libraries or ribosome display libraries. Phage display is Petition 870250078518, dated 03 / 09 / 2025, page 62 / 222 44 / 188 described, for example, in Ladner et al., Patent Document No. US 5,223,409; Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

[0081] In addition to the use of display libraries, the relevant antigen can be used to immunize a non-human animal, for example, a rodent (such as a mouse, hamster, rabbit, or rat). In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, it is possible to genetically modify mouse strains deficient in the production of mouse antibodies with large fragments of human Ig (immunoglobulin) loci. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with the desired specificity can be produced and selected. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96 / 34096 and WO 96 / 33735.

[0082] A monoclonal antibody can also be obtained from a non-human animal and then modified, for example, humanized, deimmunized, made chimeric, etc., using recombinant DNA techniques known in the art. Examples of modified antigen-binding molecules include humanized variants of non-human antibodies, matured affinity antibodies (see, for example, Hawkins et al., J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)) and antibody mutants with altered effector functions (see, for example, US Patent Document No. 5,648,260, Kontermann and Dübel (2010), loc. cit. and Little (2009), loc. cit.). Petition 870250078518, dated 03 / 09 / 2025, page 63 / 222 45 / 188

[0083] In immunology, affinity maturation is the process by which B cells produce antibodies with increased affinity for the antigen during the course of an immune response. With repeated exposures to the same antigen, a host will produce antibodies of successively higher affinities. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antigen-binding molecules, and antibody fragments. Random mutations within CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. Additionally, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range.

[0084] A preferred type of amino acid substitution variant of antigen-binding molecules involves replacing one or more residues of the hypervariable region of an original antibody (e.g., a humanized or human antibody). In general, the resulting variant(s) selected for further development will have improved biological properties compared to the original antibody from which it / they are generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, multiple sides of the hypervariable region (e.g., 6-7 sides) are mutated to generate all possible amino acid substitutions on each side. The Petition 870250078518, dated 03 / 09 / 2025, page 64 / 222 46 / 188 antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene product III packaged in each particle. The variants displayed on phages are then screened for their biological activity (e.g., binding affinity) as disclosed in this document. In order to identify candidate sides of the hypervariable region for modification, alanine screening mutagenesis can be performed to identify residues of the hypervariable region contributing significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify points of contact between the binding domain and, for example, CS1, BCMA, CD20, CD22, FLT3, CD123, CDH3, MSLN, CLL1, or human EpCAM.These contact residues and neighboring residues are candidates for replacement according to the techniques elaborated in this document. Once such variants are generated, the variant panel is subjected to screening as described in this document, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0085] The monoclonal antibodies and antigen-binding molecules of the present invention specifically 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(s) is / are identical to or homologous to sequences Petition 870250078518, dated 03 / 09 / 2025, p. 65 / 222 47 / 188 corresponding antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US Patent Document No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The chimeric antibodies of interest in this document 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. A variety of approaches to the production of chimeric antibodies have been described. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., US Patent Document No. 4,816,567; Boss et al., Patent Document No. US 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494 and GB 2177096.

[0086] An antibody, antigen-binding molecule, antibody fragment, or antibody variant can also be modified by specific deletion of human T-cell epitopes (a method called deimmunization) by the methods disclosed, for example, in patent documents WO 98 / 52976 or WO 00 / 34317. Briefly, the variable domains of the heavy and light chains 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). For the detection of potential T-cell epitopes, a computer modeling approach called peptide chaining can be applied and, additionally, a database of binding peptides can be used. Petition 870250078518, dated 03 / 09 / 2025, page 66 / 222 48 / 188 Human MHC class II can be searched for motifs present in the VH and VL sequences, as described in patent documents WO 98 / 52976 and WO 00 / 34317. These motifs bind to any of the 18 major DR allotypes of MHC class II and thus constitute potential T cell epitopes. The detected potential T cell epitopes can be eliminated by substituting small numbers of amino acid residues in the variable domains or, preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Frequently, but not exclusively, an amino acid common to a position in human germline antibody sequences can be used. Human germline sequences are revealed, 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 V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a human sequence source, for example, for structural regions and CDRs. Consensus human structural regions can also be used, for example, as described in US Patent Document No. 6,300,064.

[0087] Humanized antibodies, antigen-binding molecules, their variants or fragments (such as Fv, Fab, Fab', F(ab')2 or other antibody antigen-binding subsequences) are antibodies or immunoglobulins of predominantly human sequences, which contain (a) Petition 870250078518, dated 03 / 09 / 2025, page 67 / 222 49 / 188 minimal sequence(s) derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues from a hypervariable region (also CDR) of the receptor are replaced by residues from a hypervariable region of a non-human species (e.g., rodent) (donor antibody) such as mouse, rat, hamster, or rabbit having the desired specificity, affinity, and capacity. In some cases, residues from the structural region (FR) of Fv of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the “humanized antibodies” as used herein may also comprise residues that are not found in either the receptor antibody or the donor antibody. These modifications are made to further refine and optimize the performance of the antibodies.The humanized antibody may also comprise at least a portion of an immunoglobulin (Fc) constant region, typically that of a human immunoglobulin. For more 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).

[0088] Humanized antibodies or their fragments can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences of human Fv variable domains. Exemplary methods for generating humanized antibodies or their fragments are provided by Morrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques 4:214; and by US 5,585,089; US 5,693,761; Petition 870250078518, dated 03 / 09 / 2025, page 68 / 222 50 / 188 US 5,693,762; US 5,859,205 and US 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of the Fv variable domains of immunoglobulin from at least one heavy or light chain. Such nucleic acids can be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0089] Humanized antibodies can also be produced using transgenic animals such as mice that express human heavy and light chain genes but are unable to express 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 (US Patent Document No. 5,225,539). All CDRs of a particular human antibody can be replaced by at least a portion of a non-human CDR, or only some of the CDRs can be replaced by non-human CDRs. It is only necessary to replace the number of CDRs required for the humanized antibody to bind to a predetermined antigen.

[0090] A humanized antibody can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or retromutations. Such altered immunoglobulin molecules can be prepared by any of several techniques known in the art (e.g., Teng et al., Petition 870250078518, dated 03 / 09 / 2025, p. 69 / 222 51 / 188 Proc. Natl. Academic. 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 239 400).

[0091] The terms human antibody, “human antigen-binding molecule” and “human binding domain” include antibodies, antigen-binding molecules and binding domains having antibody regions 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) (loc. cit.). The human antibodies, antigen-binding molecules 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 CDRs and, in particular, in CDR3.Human antibodies, antigen-binding molecules, or binding domains may have at least one, two, three, four, five, or more positions substituted by an amino acid residue that is not encoded by the human germline immunoglobulin sequence. The definition of human antibodies, antibody constructs, and binding domains as used in this document also includes fully human antibodies, which include only non-artificially and / or genetically altered human antibody sequences, as these may be derived using technologies or systems such as Xenomouse. Preferably, a "fully human antibody" does not include non-coding amino acid residues. Petition 870250078518, dated 03 / 09 / 2025, page 70 / 222 52 / 188 by human germline immunoglobulin sequences.

[0092] In some embodiments, the antigen-binding molecules of the invention are isolated or substantially pure antigen-binding molecules. Isolated or substantially pure, when used to describe the antigen-binding molecules disclosed herein, means an antigen-binding molecule that has been identified, separated and / or recovered from a component of its production environment. Preferably, the antigen-binding molecule is free or substantially free from association with all other components of its production environment. Contaminating components of its production environment, such as those resulting from recombinant transfected cells, are materials that would typically interfere with the diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.Antigen-binding molecules may, for example, constitute at least about 5%, or at least about 50%, by weight of the total protein in a given sample. It is understood that the isolated protein may constitute from 5% to 99.9% by weight of the total protein content, depending on the circumstances. The polypeptide may be prepared at a significantly higher concentration through the use of an inducible promoter or high-expression promoter, such that it is prepared at increased concentration levels. The definition includes the production of an antigen-binding molecule in a wide variety of organisms and / or host cells that are known to be present in the field. Petition 870250078518, dated 03 / 09 / 2025, p. 71 / 222 53 / 188 technique. In preferred embodiments, the antigen-binding molecule will be purified (1) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a rotary cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Typically, however, an isolated antigen-binding molecule will be prepared by at least one purification step.

[0093] The term “binding domain” characterizes in conjunction with the present invention a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target side on target molecules (antigens), for example, CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN or EpCAM and CD3, respectively. The structure and function of typically the first and third or second and fourth binding domains (recognizing, for example, CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN or EpCAM) and, preferably, also the structure and / or function of the effector binding domain (typically the second and fourth or first and third binding domains recognizing CD3), is / are based on the structure and / or function of an antibody, for example, of a full-length or whole immunoglobulin molecule and / or is / are designed from the domains of the variable light chain (VL) of an antibody or variable heavy chain (VH) or its fragment.Preferably, the antigen-binding domain(s) on the surface of target cells is / are characterized by the presence of three CDRs of the light chain (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three CDRs of the... Petition 870250078518, dated 03 / 09 / 2025, page 72 / 222 54 / 188 heavy chain (i.e., CDR1, CDR2, and CDR3 of the VH region). The effector binding domain (typically CD3) also preferably comprises the minimum structural requirements of an antibody that allow binding to the target. More preferably, the second binding domain comprises at least three CDRs of the light chain (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three CDRs of the heavy chain (i.e., CDR1, CDR2, and CDR3 of the VH region). It is anticipated that the first and / or second binding domains will be produced by or obtainable by phage display or library screening methods rather than by grafting CDR sequences from a pre-existing (monoclonal) antibody onto a template.

[0094] According to the present invention, the binding domains are in the form of one or more polypeptides. Such polypeptides may include proteinaceous and non-proteinaceous parts (e.g., chemical linkers or chemical crosslinking agents such as glutaraldehyde). The proteins (including their fragments, preferably biologically active fragments and peptides, usually having fewer than 30 amino acids) comprise two or more amino acids coupled together through a covalent peptide bond (resulting in an amino acid chain).

[0095] The term polypeptide as used in this document describes a group of molecules, usually consisting of more than 30 amino acids. Polypeptides may additionally form multimers such as dimers, trimers, and higher oligomers, i.e., consisting of more than one polypeptide molecule. The polypeptide molecules that form these dimers, trimers, etc. may Petition 870250078518, dated 03 / 09 / 2025, page 73 / 222 55 / 188 or not be identical. The corresponding higher-order structures of such multimers are consequently termed homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms peptide, polypeptide, and protein also refer to naturally modified peptides / polypeptides / proteins, wherein the modification is effected, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, and the like. A peptide, polypeptide, or protein as referred to herein may also be chemically modified as pegylated. These modifications are well known in the art and described below in this document.

[0096] Preferably, the binding domains that bind to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, CDH3, MSLN, and EpCAM and / or the binding domains that bind to CD3e are human binding domains. Antibodies and antigen-binding molecules comprising at least one human binding domain avoid some of the problems associated with antibodies or antigen-binding molecules that possess non-human variable and / or constant regions such as rodent (e.g., murine, rat, hamster, or rabbit). The presence of such rodent-derived proteins may lead to rapid elimination of antibodies or antigen-binding molecules or may lead to the generation of an immune response against the antibody or antigen-binding molecule by a patient. In order to avoid the use of Petition 870250078518, dated 03 / 09 / 2025, p. 74 / 222 56 / 188 antibodies or antigen-binding molecules derived from rodents, human or fully human antibodies / antigen-binding molecules can be generated by introducing human antibody function into a rodent such that the rodent produces fully human antibodies.

[0097] The term high molecular weight species (HMWS) typically refers to product-related variants of the bispecific molecules of the invention. HMWS may include dimers, trimers, tetramers, etc., formed from monomers that may be covalently or non-covalently linked. HMWS may consist of misfolded monomers, in which the monomer surfaces are exposed, which would not normally be the case in monomeric form. They may or may not have an impact on safety and / or efficacy, but should generally be avoided from a regulatory perspective. Typically, HMWS in the context of the present invention are determined by Size Exclusion Chromatography (SEUHPLC) and may represent a different fraction of the main peak, i.e., the desired monomeric product.

[0098] The term amino acid or amino acid residue typically refers to an amino acid whose definition is recognized in the art 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 (Gin or Q); glutamic acid (Giu or E); glycine (Giy or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Vai or V), although Petition 870250078518, dated 03 / 09 / 2025, p. 75 / 222 57 / 188 modified, synthetic, or rare amino acids may be used as desired. In general, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, VaI); a negatively charged side chain (e.g., Asp, Giu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gin, Giy, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0099] Amino acid modifications include, for example, deletions of, and / or insertions of, and / or substitutions of, residues in the amino acid sequences of antibody constructs. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processes of antibody constructs, such as changing the number or position of glycosylation sites.

[0100] For example, 1, 2, 3, 4, 5, or 6 amino acids can be inserted, replaced, or deleted in each of the CDRs (obviously, depending 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 can be inserted, replaced, or deleted in each of the FRs. Preferably, amino acid sequence insertions in the antibody construct include amino- and / or carboxyl-terminal fusions in the length range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues relative to polypeptides containing one hundred or more residues, as well as insertions between sequences of single or multiple amino acid residues. Petition 870250078518, dated 03 / 09 / 2025, page 76 / 222 58 / 188 modifications can also be made within the third domain of the antibody construct of the invention. One variant of insertion of the antibody construct of the invention includes fusing an enzyme to the N-terminus or C-terminus of the antibody construct or fusing it to a polypeptide.

[0101] The sites of greatest interest for substitution mutagenesis include (but are not limited to) heavy and / or light chain CDRs, particularly hypervariable regions, but FR alterations in the heavy and / or light chain are also contemplated. Substitutions are preferably conservative substitutions as described in this document. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids may be substituted in a CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be substituted in structural regions (FRs), depending on the length of the CDR or FR. For example, if a CDR sequence includes 6 amino acids, one, two, or three of those amino acids are predicted to be substituted. Similarly, if a CDR sequence includes 15 amino acids, one, two, three, four, five, or six of those amino acids are predicted to be substituted.

[0102] A useful method for identifying certain residues or regions of antibody constructs that are preferential sites for mutagenesis is called alanine-scavenging mutagenesis as described by Cunningham and Wells in Science, 244: 1081-1085 (1989). Here, a target residue or group of residues within the antibody construct is / are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with an amino acid. Petition 870250078518, dated 03 / 09 / 2025, page 77 / 222 59 / 188 neutral or negatively charged (with maximum preference for alanine or polyalanine) to affect the interaction of amino acids with the epitope.

[0103] These amino acid locations demonstrating functional sensitivity to substitutions are then refined by introducing additional variants or others at, or to, the substitution sites. Thus, while the location or region to introduce a variation of amino acid sequences is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze or optimize the performance of a mutation at a given location, alanine-tracking or random mutagenesis can be conducted at a 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 locations in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis.Screening for mutants is done using antigen-binding activity assays, such as target cell surface antigen binding or CD3.

[0104] In general, if amino acids are substituted in one or more or in all of the heavy and / or light chain CDRs, it is preferable that the substituted sequence thus obtained be at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% to 95% identical to the original CDR sequence. This means that the degree to which the CDR is identical to the substituted sequence depends on its length. For example, a CDR having 5 amino acids is Petition 870250078518, dated 03 / 09 / 2025, p. 78 / 222 60 / 188 preferably 80% identical to its substituted sequence so as to have at least one substituted amino acid. Consequently, the CDRs of the antibody construct may have different degrees of identity with respect to their substituted sequences, for example, CDRL1 may be 80%, while CDRL3 may be 90%.

[0105] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitution or one or more of the “exemplary substitutions” listed in Table 3 below) is permitted provided that the antibody construct retains its ability to bind to the target cell surface antigen through the first binding domain and to CD3, respectively, CD3 epsilon through the second domain and / or its CDRs have an identity with respect to the sequence then substituted (at least 60% or 65%, more preferably 70% to 75%, even more preferably 80% to 85%, and particularly, preferably, 90% to 95% identical to the “original” CDR sequence).

[0106] Conservative substitutions are shown in Table 3 under the heading “preferred substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, termed “exemplary substitutions” in Table 3, or as further described below in reference to amino acid classes, may be introduced and the products screened for a desired characteristic. Table 1: Amino acid substitutions Original Substitutions Exemplary Substitutions Preferred Substitutions Petition 870250078518, dated 03 / 09 / 2025, page 79 / 222 61 / 188 Ala (A) val, leu, ile Val Arg I lys, gln, asn Lys Asn (N) gln, his, asp, lys, arg Gln Asp (D) glu, asn Glu Cys I ser, ala Ser Gln (Q) asn, glu Asn Glu I asp, gln Asp Gly (G) Ala Ala His (H) asn, gln, lys, arg Arg Ile (I) leu, val, met, ala, phe Leu Leu (L) norleucine, ile, val, met, ala Ile Lys (K) arg, gln, asn Arg Met (M) leu, phe, ile Leu Phe (F) leu, val, ile, ala, tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) tyr, phe Tyr Tyr (Y) trp, phe, thr, be Phe Val (V) ile, leu, met, phe, ala Leu

[0107] Substantial modifications in the biological properties of the antibody construct of the present invention are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the Petition 870250078518, dated 03 / 09 / 2025, page 80 / 222 62 / 188 structure of the polypeptide backbone in the area of ​​the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0108] Non-conservative substitutions will involve replacing a member of one of these classes with another class. Any cysteine ​​residue not involved in maintaining the appropriate conformation of the antibody construct can be substituted, usually with serine, to enhance the oxidative stability of the molecule and avoid anomalous crosslinking. Conversely, cysteine ​​link(s) can be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0109] For amino acid sequences, sequence identity and / or similarity is determined using 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 similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin). Petition 870250078518, dated 03 / 09 / 2025, page 81 / 222 63 / 188 Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using the standard definitions or by inspection. Preferably, the percentage of 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 join penalty of 30, “Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0110] An example of a useful algorithm is PILEUP. PILEUP creates a multi-sequence alignment from a group of related sequences using pairwise, forward alignments. It can also graphically represent a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the forward alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a standard gap weight of 3.00, a standard gap length of 0.10, and weighted end gaps.

[0111] 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 which was obtained from Altschul et al., Petition 870250078518, dated 03 / 09 / 2025, page 82 / 222 64 / 188 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap period=1, overlap fraction=0.125, word limit (T)=II. The HSP S and HSP S2 parameters are dynamic values ​​and are set by the program itself depending on the composition of the particular sequence and the composition of the particular database against which the sequence of interest is being searched; however, the values ​​can be adjusted to increase sensitivity.

[0112] An additional useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res. 25:33893402. Gapped BLAST uses BLOSUM-62 replacement scores; threshold parameter T set to 9; the two-hit method to trigger gapless extensions charges gap lengths of k a cost of 10+k; Xu set to 16 and Xg set to 40 for the database search step and to 67 for the output step of the algorithms. Gapless alignments are triggered by a sort matching approximately 22 bits.

[0113] Generally, the homology, similarity, or identity of amino acids between individual variant CDRs or VH / VL sequences is at least 60% with the sequences illustrated in this document and, more typically, with preferably increasing homologies or identities of at least 65% or 70%, more preferably at least 75% or 80%, even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and nearly 100%. Similarly, “percent (%) of Petition 870250078518, dated 03 / 09 / 2025, p. 83 / 222 65 / 188 Nucleic acid sequence identity with respect to the nucleic acid sequence of the binding proteins identified in this document is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody construct. A specific method uses the BLASTN module of WU-BLAST-2 defined in standard parameters, with overlap period and overlap fraction set to 1 and 0.125, respectively.

[0114] In general, the homology, similarity, or identity of nucleic acid sequences between the nucleotide sequences encoding individual variant CDRs or VH / VL sequences and the nucleotide sequences illustrated in this document is at least 60% and, more typically, with homologies and identities preferably increasing to at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and nearly 100%. Thus, a variant CDR or variant VH / VL region is one with the specified homology, similarity, or identity with the original 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 parental CDR or VH / VL.

[0115] In one embodiment, the percentage of identity with respect to the human germline of the antibody constructs according to the invention is 70% or 75%, more preferably 80% or 85%, even more preferably Petition 870250078518, dated 03 / 09 / 2025, page 84 / 222 66 / 188 > 90%, and with maximum preference > 91%, > 92%, > 93%, > 94%, > 95% or even > 96%. It is thought that identity with germline gene products of human antibodies is an important characteristic to reduce the risk of therapeutic proteins provoking an immune response against the drug in the patient during treatment. Hwang & Foote (“Immunogenicity of engineered antibodies; Methods 36 (2005) 3 to 10) demonstrate that reducing non-human portions of drug antibody constructs leads to a decreased risk of inducing anti-drug antibodies in patients during treatment. By comparing an exhaustive number of clinically evaluated antibody drugs and their respective immunogenicity data, a trend is shown that humanization of antibody V regions makes the protein less immunogenic (average 5.1% of patients) than antibodies carrying unaltered non-human V regions (average 23.59% of patients).A higher degree of identity to human sequences is therefore desirable for V-region-based protein therapeutics in the form of antibody constructs. For this purpose of germline identity determination, VL regions can be aligned with the amino acid sequences of V and J segments of the human germline (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the amino acid sequence calculated by dividing the identical amino acid residues by the total number of amino acid residues in the VL as a percentage. The same can be done for VH segments (http: / / vbase.mrc-cpe.cam.ac.uk / ), with the exception that VH CDR3 can be excluded due to its high density. Petition 870250078518, dated 03 / 09 / 2025, p. 85 / 222 67 / 188 and an absence of CDR3 alignment partners from human germline VH. Recombinant techniques can then be used to increase sequence identity with human germline antibody genes.

[0116] In a further embodiment, the bispecific antibody constructs of the present 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 of supernatant, more preferably > 0.5 mg / L, even more preferably > 1 mg / L and most preferably > 3 mg / L of supernatant.

[0117] Similarly, the yield of dimeric antibody construct isoforms and consequently the percentage of monomer (i.e., monomer: (monomer+dimer)) of the antibody constructs can be determined. The productivity of monomeric and dimeric antibody constructs and the calculated percentage of monomer can, for example, be obtained in the SEC purification step from culture supernatants from standardized research-scale production in cylindrical bottles. In one embodiment, the percentage of monomer of the antibody constructs is > 80%, more preferably > 85%, even more preferably > 90%, and most preferably > 95%.

[0118] In one embodiment, antibody constructs have a preferential plasma stability (ratio of EC50 with plasma to EC50 s / c plasma) of < 5 or < 4, with greater preference < 3.5 or < 3, even more preferentially < 2.5 or < 2, and with maximum preference 1.5 or < 1. The stability Petition 870250078518, dated 03 / 09 / 2025, p. 86 / 222 A 68 / 188 plasma concentration of an antibody construct can be tested by incubating the construct in human plasma at 37°C for 24 hours followed by EC50 determination in a chromium release cytotoxicity assay. Effector cells in the cytotoxicity assay can be stimulated enriched human CD8-positive T cells. Target cells can be, for example, CHO cells transfected with human target cell surface antigen. The effector-to-target cell ratio (E:T) can be chosen as 10:1. The human plasma pooling used for this purpose is derived from healthy donor blood collected using EDTA-coated syringes. Cellular components are removed by centrifugation, and the upper plasma phase is collected and subsequently pooled. As a control, antibody constructs are diluted immediately before the cytotoxicity assay in RPMI-1640 medium.Plasma stability is calculated as a ratio between EC50 (after plasma incubation) and EC50 (control).

[0119] It is further preferred that the monomer-to-dimer conversion of antibody constructs of the invention be small. The conversion can be measured under different conditions and analyzed by high-performance size exclusion chromatography. For example, the incubation of the monomeric isoforms of the antibody constructs can be carried out for 7 days at 37 °C and concentrations of, for example, 100 μg / mL or 250 μg / mL in an incubator. Under these conditions, it is preferred that the antibody constructs of the invention show a dimer percentage that is <5%, more preferably <4%, even more preferably <3%, even more preferably <2.5%, even more preferably <2 Petition 870250078518, dated 03 / 09 / 2025, page 87 / 222 69 / 188%, with even greater preference <1.5% and maximum preference <1% or <0.5% or even 0%.

[0120] It is also preferable that the bispecific antibody constructs of the present invention exhibit very low dimer conversion after numerous freeze / thaw cycles. For example, the antibody construct monomer is adjusted to a concentration of 250 μg / mL, for example, in generic formulation buffer and subjected to freeze / thaw cycles (freezing at -80 °C for 30 min followed by thawing for 30 min at room temperature), followed by high-performance SEC to determine the percentage of initially monomeric antibody construct that has been converted into dimeric antibody construct.Preferably, the dimer percentages of the bispecific antibody constructs are <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.

[0121] The bispecific antibody constructs of the present invention preferably exhibit favorable thermostability at aggregation temperatures above k45 °C or k50 °C, more preferably k52 °C or k54 °C, even more preferably k56 °C or k57 °C, and most preferably k58 °C or k59 °C. The thermostability parameter can be determined in terms of antibody aggregation temperature as follows: Antibody solution at a concentration of 250 pg / mL is transferred to a single-use cuvette and placed in Petition 870250078518, dated 03 / 09 / 2025, page 88 / 222 70 / 188 Dynamic Light Scattering (DLS) device. The sample is heated from 40 °C to 70 °C at a heating rate of 0.5 °C / min with constant acquisition of the measured radius. The increase in radius indicating protein melting and aggregation is used to calculate the antibody aggregation temperature.

[0122] Alternatively, temperature melting curves can be determined by differential scanning calorimetry (DSC) to determine intrinsic biophysical protein stabilities of antibody constructs. These experiments are performed using a MicroCal LLC VP-DSC device (Northampton, MA, USA). The energy absorption of a sample containing an antibody construct is recorded from 20 °C to 90 °C compared to a sample containing only the formulation buffer. The antibody constructs are adjusted to a final concentration of 250 μg / mL, for example, in SEC operating buffer. To record the respective melting curve, the overall sample temperature is increased step by step. At each temperature T, the energy absorption of the sample and the formulation buffer reference is recorded. The difference in energy absorption Cp (kcal / mol / °C) of the sample minus the reference is plotted against the respective temperature.The melting point is defined as the temperature at the first maximum of energy absorption.

[0123] It is also anticipated that the bispecific antigenxCD3 antibody constructs of the invention will have a turbidity (as measured by OD340 after concentration of purified monomeric antibody construct to 2.5 mg / mL and overnight incubation) of ^ 0.2, preferably ^ 0.15, more preferably ^ 0.12, yet Petition 870250078518, dated 03 / 09 / 2025, page 89 / 222 71 / 188 with a preference of < 0.1 and a maximum preference of < 0.08.

[0124] Furthermore, it is anticipated that the bispecific antibody constructs of the present invention will exhibit therapeutic efficacy or antitumor activity. This can, for example, be analyzed in a study as revealed in the following example of an advanced-stage human tumor xenograft model:

[0125] A person skilled in the art knows how to modify or adapt certain parameters of this study, such as the number of tumor cells injected, the injection site, the number of human T cells transplanted, the amount of bispecific antibody constructs to be administered, and the timelines, while still arriving at a significant and reproducible result. Preferably, the T / C tumor growth inhibition [%] 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.

[0126] In a preferred embodiment of the antibody construct of the invention, the antibody construct is a single-chain antibody construct.

[0127] Also in a preferred embodiment of the antibody construct of the invention, said third domain comprises an amino-to-carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0128] In another embodiment of the invention, the CH2 domain of one or preferably each (both) polypeptide monomers of the third domain comprises an intra-domain cysteine ​​disulfide bridge. As known in the art, the term cysteine ​​disulfide bridge refers to Petition 870250078518, dated 03 / 09 / 2025, pages 90 / 222 72 / 188 to a functional group with the general structure R—S—S—R. The linkage is also called an SS linkage or a disulfide bridge and is derived by the coupling of two thiol groups of cysteine ​​residues. It is particularly preferred for the antibody construct of the present invention 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).

[0129] In one embodiment of the invention, a glycosylation site at Kabat position 314 of the CH2 domain is removed. It is preferred that this removal of the glycosylation site be achieved by an N314X substitution, where X is any amino acid excluding Q. The said substitution is preferably an N314G substitution. In a more preferred embodiment, said CH2 domain further comprises the following substitutions (position according to Kabat) V321C and R309C (these substitutions introduce the intradomain cysteine ​​disulfide bridge at Kabat positions 309 and 321).

[0130] It is assumed that the preferential characteristics of the antibody construct of the invention compared, for example, with the known bispecific heteroFc antibody construct (Figure 1b) may be related, among other things, to the introduction of the modifications described above in the CH2 domain. Thus, it is preferable for the construct of the invention that the CH2 domains of the third domain of the antibody construct of the invention comprise an intra-domain cysteine ​​disulfide bridge at Kabat positions 309 and 321 and / or the glycosylation site at the Kabat position. Petition 870250078518, dated 03 / 09 / 2025, pp. 91 / 222 73 / 188 314 is removed by replacing it with an N314X as above, preferably with an N314G replacement.

[0131] In another preferred embodiment of the invention, the CH2 domains of the third domain of the antibody construct of the invention comprise the intradomain cysteine ​​disulfide bridge at Kabat positions 309 and 321 and the glycosylation site at Kabat position 314 is removed by an N314G substitution.

[0132] In one embodiment, the invention provides an antibody construct, wherein: (I) the first domain comprises two variable antibody domains and the second domain comprises two variable antibody 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 variable antibody domains and the second domain comprises one variable antibody domain; or (iv) the first domain comprises one variable antibody domain and the second domain comprises one variable antibody domain.

[0133] Consequently, the first and second domains may be binding domains each comprising two variable antibody domains such as a VH and a VL domain. Examples of such binding domains comprising two variable antibody domains as described above include, for example, Fv fragments, scFv fragments, or Fab fragments described earlier. Alternatively, one or both binding domains may comprise only one Petition 870250078518, dated 03 / 09 / 2025, page 92 / 222 74 / 188 single variable domain. Examples of such single-domain binding domains, as described above in this document, include, for example, single variable domain nanobodies or antibodies comprising only one variable domain, which may be VHH, VH or VL, that bind specifically to an antigen or epitope independently of other V regions or domains.

[0134] In a preferred embodiment of the antibody construct of the present invention, the first and second domains are fused with the third domain via a peptide linker. The preferred peptide linker has been described above in this document and is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187) or its polymers, i.e., (Gly4Ser)x, wherein x is an integer of 1 or greater (e.g., 2 or 3). A particularly preferred linker for fusing the first and second domains with the third domain is represented in SEQ ID NOs: 1.

[0135] In a preferred embodiment, the antibody construct of the invention is characterized by comprising, in an amino-to-carboxyl terminal order: (a) the first domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-189; (c) the second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of the following SEQ ID NOs: 187, 188, 189, 195, 196, 197 and 198; (e) the first polypeptide monomer of the third domain; Petition 870250078518, dated 03 / 09 / 2025, page 93 / 222 75 / 188 (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) the second polypeptide monomer of the third domain.

[0136] In one aspect of the invention, the target cell surface antigen bound by the first domain is a tumor antigen, an antigen specific for an immune disorder, or a viral antigen. The term tumor antigen as used herein may be understood as those antigens that are presented on tumor cells. These antigens may be presented on the cell surface with an extracellular portion, which is often combined with a transmembrane and cytoplasmic portion of the molecule. These antigens may sometimes be presented only by tumor cells and never by normal cells.Tumor antigens may be exclusively expressed in tumor cells or may represent a tumor-specific mutation compared to normal cells. In this case, they are called tumor-specific antigens. More common are antigens that are presented by both tumor cells and normal cells and are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells or are 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 in this document, are CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, BCMA, and PSMA. Petition 870250078518, dated 03 / 09 / 2025, page 94 / 222 76 / 188

[0137] In a preferred embodiment of the antibody construct of the invention, the tumor antigen is preferably selected from the group consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, BCMA and PSMA.

[0138] In one aspect of the invention, the antibody construct comprises an amino-to-carboxyl order: (a) the first domain having an amino acid sequence selected from the group consisting of the following 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, 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, 181, 223, 235 and 246, (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-189; (c) the 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 of SEQ ID NO: 202; (d) a peptide linker having an amino acid sequence selected from the group consisting of the following SEQ ID NOs: 187, 188, 189, 195, 196, 197 and 198; (e) the first polypeptide monomer of the third domain having a polypeptide sequence selected from the group consisting of SEQ IDs 249-256; Petition 870250078518, dated 03 / 09 / 2025, page 95 / 222 77 / 188 (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) the second polypeptide monomer of the third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 249-256.

[0139] In one aspect, the bispecific antibody construct of the invention is characterized by having an amino acid sequence selected from the group consisting of and being directed to the respective target cell surface antigen: (a) (b) SEQ SEQ ID ID NOs: 27, 28, NOs: each from 37 to 48 to 41; 52; CD33 EGFRvIII (c) SEQ ID NOs: each from 59 to 64; MSLN (d) SEQ ID NOs: each from 71 to 82 CDH19 (e) SEQ ID NOs: each from 100 to 104 DLL3 (f) SEQ ID NOs: 7, 8, 17, 113 and 114 CD19 (g) SEQ ID NOs: each from 89 to 93 FLT3 (h) SEQ ID NOs: each from 121 to 125 CDH3 (i) SEQ ID NOs: each from 132 to 136 BCMA (j) SEQ ID NOs: each from 143 to 151, 158 to 166 and 173 to 181 PSMA (k) SEQ ID NO 213 MUC17 (l) SEQ ID NOs: each from 225 and 237 CLDN18, and (m) SEQ ID No: 248 CD70

[0140] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to DLL3 comprising a variable anti-DLL3 light domain comprising. Petition 870250078518, dated 03 / 09 / 2025, pp. 96 / 222 78 / 188

[0141] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to BCMA, which comprises a variable anti-BCMA light domain.

[0142] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to CD33 which comprises a variable anti-CD33 light domain.

[0143] In some aspects of the revelation, the protein, as an antibody or bispecific antibody construct, is present in the liquid formulation (before lyophilization) in an amount ranging from about 0.1 mg / mL to about 100 mg / mL (or about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL) or 100 mg / mL). For example, the protein is optionally present in the liquid formulation at a concentration ranging from about 0.1 mg / mL to about 70 mg / mL.In some cases, the protein is present in the liquid formulation in an amount ranging from about 0.5 mg / mL to about 30 mg / mL (or about 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL or 30 mg / mL). In several cases, the protein is present in the liquid formulation in an amount ranging from about 1 mg / mL to about 20 mg / mL (or about 1 mg / mL, 1.5 mg / mL, 2 mg / mL, etc.). Petition 870250078518, dated 03 / 09 / 2025, page 97 / 222 79 / 188 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 16 mg / mL, 16.5 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.5 mg / mL, 19 mg / mL, 19.5 mg / mL or 20 mg / mL). In some respects, the protein is present in the liquid formulation at a concentration of approximately 21 mg / mL.

[0144] The protein formulation of the development optionally comprises a saccharide. In some embodiments, the saccharide is a monosaccharide or a disaccharide. Suitable saccharides include, for example, glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof. In some cases, the saccharide comprises trehalose.

[0145] In some aspects, the liquid formulation (before lyophilization) comprises saccharide at a concentration of about 1% to about 15% w / v or about 4% to about 13% w / v or about 6% to about 12% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13% or at least 14% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about Petition 870250078518, dated 03 / 09 / 2025, p. 98 / 222 80 / 188% or about 15% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5% or about 12% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of about 7% to about 12% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of about 9% w / v. In some embodiments, the saccharide is sucrose and is present in the liquid formulation at a concentration ranging from about 6% to about 12% w / v.

[0146] The protein formulation of the development optionally comprises a surfactant. Suitable surfactants include a polysorbate, a poloxomer, a polyoxyethylene, or any combination thereof. Surfactants contemplated include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X100, polyoxyethylene, PEG 3350, PEG 4000, and any combination thereof. In some respects, the surfactant comprises a polysorbate. In some cases, the surfactant is polysorbate 20.

[0147] The protein formulations described in this document may comprise a surfactant or a mixture of surfactants (although this is not required). In some respects, the liquid formulation (before lyophilization) comprises a surfactant at a concentration of about 0.001% to about 5% w / v (or about 0.001% to about 0.5% or about 0.004% to about 0.5% w / v or about 0.001% to about 0.01% w / v or about 0.004% to about 0.01% w / v). Petition 870250078518, dated 03 / 09 / 2025, page 99 / 222 81 / 188 In some aspects, the liquid formulation comprises a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0 or at least 4.5% w / v. In some aspects, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.5% w / v. In some aspects, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some aspects, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some aspects, the liquid formulation comprises a surfactant at a concentration of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, to about 0.5% w / v.In some respects, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01% w / v.

[0148] The protein formulation of the development optionally comprises a buffer. Suitable buffers include acetate buffers, glutamate buffers, citrate buffers, lactate buffers, succinate buffers, tartrate buffers, fumarate buffers, maleate buffers, histidine buffers, phosphate buffers, 2-(N-morpholino)ethanesulfonate buffers or any Petition 870250078518, dated 03 / 09 / 2025, pages 100 / 222 82 / 188 its combination. In some cases, the buffer comprises histidine.

[0149] Buffering agents are frequently employed to control the pH in the formulation. In some cases, the buffer is added at a concentration that maintains the pH of the liquid formulation from about 3 to about 7, or about 4 to about 6, about 4 to 5, or about 5 to about 6, or about 6 to about 6.5. The effect of pH on formulations can be characterized using any one or more of several approaches such as accelerated stability studies and calorimetric tracking studies (Remmele RL Jr., et al., Biochemistry, 38 (16): 5241-7 (1999)).

[0150] The buffer system (when present in the protein formulation) is selected to be physiologically compatible and maintain a desired pH. The buffer may be present in the liquid formulation (before lyophilization) at a concentration between about 0.1 mM and about 1000 mM (1 M) or between about 5 mM and about 200 mM or between about 5 mM and about 100 mM or between about 10 mM and about 50 mM. Suitable buffer concentrations encompass concentrations of about 200 mM or less. In some respects, the buffer in the liquid protein formulation (before lyophilization) is present at a concentration of approximately 190 mM, approximately 180 mM, approximately 170 mM, approximately 160 mM, approximately 150 mM, approximately 140 mM, approximately 130 mM, approximately 120 mM, approximately 110 mM, approximately 100 mM, approximately 80 mM, approximately 70 mM, approximately 60 mM, approximately 50 mM, approximately 40 mM, approximately 30 mM, approximately 20 mM, approximately 10 mM, or approximately 5 mM.In some respects, the buffer concentration is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4. Petition 870250078518, dated 03 / 09 / 2025, pages 101 / 222 83 / 188 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700 or 900 mM. In some respects, the buffer concentration is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80 or 90 mM and 100 mM. In some aspects, the buffer concentration is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30 or 40 mM and 50 mM.

[0151] As a further aspect, kits are provided comprising a lyophilized protein composition described herein, packaged in a manner that facilitates administration to individuals. In one aspect, the kit includes a lyophilized protein composition described herein, packaged in a container, such as a sealed vial, container, single-use or multi-use vial, pre-filled device (e.g., syringe) or pre-filled injection device, optionally with a label affixed to the container or included in the packaging describing the use of the lyophilized protein composition. In one aspect, the pharmaceutical composition is packaged in a unit dosage form. The kit may include a device suitable for administering the reconstituted protein composition according to a specific route of administration, although this is not required.For example, the disclosure provides a dual-chamber device to deliver a reconstituted protein composition disclosed in this document to a subject in need thereof. Dual-chamber devices are combined products containing the lyophilized protein composition disclosed in this document and a diluent in two separate chambers of the device. Pre-devices. Petition 870250078518, dated 03 / 09 / 2025, pp. 102 / 222 84 / 188 filled dual-chamber devices are combination products that contain lyophilized drug and diluent in two separate chambers of the device. Dual-chamber devices suitable for use with the present disclosure are described in the art. See, for example, Ingle R., Fang W. (2021). Int. Journal of Pharmaceuticals 597, 12031.

[0152] The formulation described in this document is useful as a pharmaceutical formulation in the treatment or improvement of cancer in a subject in need thereof. The terms “subject in need” or “those in need of treatment” include subjects already suffering from the disorder, as well as those in whom the disorder should be prevented. The “subject in need” or “patient” includes human subjects and other mammals receiving prophylactic or therapeutic treatment. “Treatment” does not require complete remission or eradication of the disease; any improvement of the disease and / or improvement of symptoms associated with the disease are contemplated.For example, a therapeutic response will refer to one or more of the following improvements in the disease: (1) reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing down to some extent, preferably stopping) of tumor growth or the appearance of new lesions; (5) slowing of disease progression; (6) an increased patient survival rate; (7) downgrading of a cancer stage (e.g., Stage 2 to Stage 1); and / or (8) some relief of one or more symptoms associated with the disease or condition. The composition may also be administered to prevent diseases or delay their onset, for example, to prevent a. Petition 870250078518, dated 03 / 09 / 2025, pages 103 / 222 85 / 188 occurrence or recurrence of a tumor or cancer. The disease status is monitored by, for example, clinical examination, X-ray, computed tomography (CT, such as spiral CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels (e.g., carcinoembryonic antigen (CEA)), cytology, histology, tumor biopsy sampling, and / or circulating tumor cell count. These methods are also typically used to diagnose and stage cancer.

[0153] The disclosure provides a method of treating cancer, comprising administering to a subject in need a therapeutically effective amount of a reconstituted composition based on the lyophilized formulation described herein. In certain embodiments, the subject is a human being. In certain aspects, the cancer is a solid tumor.In some forms, the cancer is brain cancer, bladder cancer, breast cancer (e.g., triple-negative breast cancer), clear cell kidney cancer, cervical cancer, colon and rectal cancer, endometrial cancer, gastric cancer, squamous cell carcinoma of the head / neck, lip and mouth cancer, liver cancer, squamous cell carcinoma of the lung, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, oral cancer, pancreatic cancer, prostate cancer, neuroendocrine prostate cancer, renal cell carcinoma, sarcoma, small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck (HNSCC), or thyroid cancer. In some forms, the cancer is acute lymphoblastic leukemia. Petition 870250078518, dated 03 / 09 / 2025, pp. 104 / 222 86 / 188 (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). In some aspects, the cancer is diffuse large B-cell lymphoma (DLBCB), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL). The disclosure also provides for the use of a reconstituted composition based on the lyophilized formulation in a cancer treatment method, as well as the use of the lyophilized formulation in the preparation of a drug to treat cancer.

[0154] Preferably, the pharmaceutical formulation is administered parenterally, for example, intravenously, subcutaneously, intratumorally, or intramuscularly. Parenteral administration can be performed by injection, such as a bolus injection, or by infusion, such as a continuous infusion. Administration can be performed via depot for long-term release. In some embodiments, the formulation is administered intravenously by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of the pharmaceutical product. In some embodiments, the formulation is administered as a single dose. Pharmaceutical formulations can be administered using a medical device. Examples of medical devices for administering pharmaceutical formulations are described in US patent documents No. 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. Petition 870250078518, dated 03 / 09 / 2025, pp. 105 / 222 87 / 188

[0155] The examples below illustrate representative features of the disclosure. From the description of these aspects, other aspects of the invention can be made and / or practiced based on the description provided below. The methods involve the use of molecular biological techniques described in treatises such as Molecular Cloning: A Laboratory Handbook, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. The example serves only to illustrate the invention and is not intended to limit the scope of the invention in any way. EXAMPLE

[0156] This example describes a process for preparing a lyophilized bispecific molecule composition according to an alternative method of including an annealing step and according to the present invention of including a CIN step, the latter resulting in a desired product property of low percent HMWS and desirable process time.

[0157] The use of annealing and controlled ice nucleation are two ways to address the challenges associated with freeze-drying, including aggregation and long processing times. Annealing and controlled ice nucleation solve this problem by reducing ice heterogeneity by producing larger ice crystals, resulting in a porous cake matrix with less resistance to mass transfer during sublimation.

[0158] Annealing involves maintaining the product between the glass transition and eutectic melting temperatures after Petition 870250078518, dated 03 / 09 / 2025, pp. 106 / 222 88 / 188 Stochastic ice nucleation (Figure 1A). This melts the ice crystals, of which the smaller ones disappear completely, leaving the larger ones behind. Subsequent freezing causes the larger crystals to grow even larger. Larger ice crystals lead to larger pores in the freeze-dried cake, which corresponds to less resistance to vapor moving through the dry layer and faster primary drying. However, maintaining the product above the glass transition temperature can promote the formation of high molecular weight species (HMWS) for some products due to increased mobility and proximity of proteins. This problem has been observed in products with bispecific molecules (e.g., BiTE® molecule) with protein concentrations above 5 mg / mL. Additionally, annealing prolongs the freeze-drying phase, which partially compensates for the reduction in primary drying time.

[0159] Controlled ice nucleation (CIN) produces larger ice crystals using a different method. For example, a CIN technique involves injecting seed crystals to trigger ice nucleation (Figure 1B). This triggers nucleation while the samples are only slightly supercooled, and an isothermal hold after nucleation can be used to make the crystals grow further. The lower degree of supercooling results in larger ice crystals than those created in stochastic nucleation, and a shorter isothermal hold may represent a lower risk of HMWS formation when compared to annealing. In this example, a freeze-drying cycle of engaging molecule of Petition 870250078518, dated 03 / 09 / 2025, pages 107 / 222 89 / 188 bispecific single-chain T cells without a CIN step, with and without annealing, were compared with a CIN lyophilization to evaluate bispecific molecule drugs with “high protein concentration” (defined in this example as 15 mg / mL or more). The bispecific molecules BCMAxCD3 (20 mg / mL) and DLL3xCD3 (15 mg / mL) were used as high-concentration bispecific molecule products. Currently, annealing is a typical option for lyophilized bispecific molecule drugs. Thus, such a lyophilization cycle was used as the representative annealing lyophilization cycle in this document. The annealing step was removed from a lyophilization cycle to create a “no-annealing” cycle. The CIN lyophilization cycle, initially developed for the bispecific molecule BCMAxCD3 1 mg 6R SKU7, has been adapted for use with high-concentration bispecific molecule formulations.The freeze-drying cycles were evaluated in terms of total cycle time and product quality. Materials and methods

[0160] The bispecific molecule BCMAxCD3 (20 mg / mL) and the bispecific molecule DLL3xCD3 (15 mg / mL) were used for these experiments. Table 1 contains details of the drug substances (DS) used. Table 1. Bispecific Molecular Drug Substances Material Formulation: Molecule 20 mg / mL in bispecific L-glutamate, 9% w / v BCMAxCD3 of sucrose, 0.01% Petition 870250078518, dated 03 / 09 / 2025, pages 108 / 222 90 / 188 w / v polysorbate 80, pH 4.2. Molecule 15 mg / mL in L-specific glutamate, 9% w / v DLL3xCD3 sucrose, 0.01% w / v polysorbate 80, pH 4.2

[0161] The bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3 DS were stored at -70 °C and -30 °C, respectively. Before use, the DS was thawed at room temperature for 72 hours. 150 mL aliquots were collected and mixed by inversion for 2 min. The DS was then filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter before filling vials.

[0162] Table 2 and Table 3 cover the equipment and components of the flasks used in this study. The 6R flasks were depyrogenated before use. The sterilized stoppers were stored in autoclaved packaging before use, but without maintaining the sterile seal after opening the packaging. Table 2. Equipment used in experiments Equipment Manufacturer Millrock REVO Pro Lyo Unit Millrock Repeater Pipette Brandtech Scientific Karl Fischer Moisture Analyzer Metrohm HIAC Particle Counter Hach Petition 870250078518, dated 03 / 09 / 2025, pp. 109 / 222 91 / 188 HPLC Agilent Technologies, Waters Instruments Mettler Toledo pH Meter Osmometer Advanced Instruments Spectrophotometer Agilent Technologies Table 3. Bottle components Component Bottle, 6R Freeze-drying stopper, 20 mm Seal, 20 mm Summary of Experiments

[0163] The purpose of these experiments was to compare the impact of a conventional lyophilization cycle with and without annealing with a CIN lyophilization cycle for the high-concentration model bispecific molecule BCMAxCD3 (20 mg / mL) and the bispecific molecule DLL3xCD3 (15 mg / mL). A lyophilization cycle of bispecific molecules was used as the conventional lyophilization cycle with and without annealing. The CIN lyophilization cycle was based on the previous CIN development for the bispecific molecule BCMAxCD3 1 mg 6R SKU7. A summary of all three lyophilization cycles can be found in Table 4. Table 4. Summary of the freeze-drying cycle. Petition 870250078518, dated 03 / 09 / 2025, pp. 110 / 222 92 / 188 Name of the freeze-drying cycle Does it use CIN? Does it use annealing? Description Cycle without CIN Standard No Yes Freeze-drying cycle for bispecific molecules Cycle without standard and without annealing No No A modification of the freeze-drying cycle for bispecific molecules with the annealing step removed. Created for comparison purposes only. CIN cycle Yes No A CIN cycle first developed for the bispecific molecule BCMAxCD3 1 mg 6R SKU Freeze-drying procedure:

[0164] Prior to filling, 6R vials were used to completely fill a Millrock lyophilization tray. A total of 100 6R vials were filled with 1.3 mL of 20 mg / mL of the bispecific molecule BCMAxCD3 DS and another 100 6R vials were filled with 1.3 mL of 15 mg / mL of the bispecific molecule DLL3xCD3 DS. Filling 1.3 mL into a 6R vial presentation was chosen as a Petition 870250078518, dated 03 / 09 / 2025, pages 111 / 222 Figure 93 / 188 shows a high-concentration representation of a BiTE, consisting of a 1.28 mL filling of bispecific molecule formulations < 5 mg / mL in a 6R vial. The two vial sets were arranged side-by-side in a single lyophilization tray. Thermocouples were placed in front of, center of, and behind the filled samples.

[0165] The freeze-drying cycle parameters for the freeze-drying cycle with and without annealing and the CIN cycle are presented in Table 5. Bispecific molecule BCMAxCD3. The primary and secondary drying parameters remain unchanged. However, the nucleation conditions were adjusted to minimize the risk of HMWS formation. This involved reducing the nucleation temperature from the less favorable -7 °C to the more favorable -12, -15 or -17 °C according to the present invention and setting the post-nucleation to 90 minutes or even reducing the holding time from one hour to 30 or 20 minutes (Figure 3). These changes created a CIN freezing phase that reduced the time the product spent in a cryoconcentrated muddy state, which reduces the chances of HMWS formation. Table 5. Lyophilization cycle parameters Petition 870250078518, dated 03 / 09 / 2025, pages 112 / 222 94 / 188 Freeze-Drying Stage Parameter Cycle without standard CIN “Cycle without standard CIN” and without annealing CIN Cycle (center) CIN Cycle (low) CIN Cycle (high) CIN Freezing Conditions: Prenucleation temperature (°C) and equilibration time (min); Postnucleation temperature (°C) and postnucleation retention time (h) -15 °C and 110 min; -15 °C and 30 min -17 °C and 90 min; -17 °C and 20 min -12 °C and 120 min; -12 °C and 90 min Freezing ramp 1 (°C / min) 0.5 °C / min 0.2 °C / min 0.2 °C / min 0.2 °C / min Petition 870250078518, dated 03 / 09 / 2025, pp. 113 / 222 95 / 188 Freeze-Drying Stage Parameter Cycle without standard CIN Cycle without standard CIN and without annealing CIN Cycle (center) CIN Cycle (low) CIN Cycle (high) Freezing temperature (°C) and retention time (h) -45 °C and 2 h -45 °C and 120 or 180 min -50 °C and 150 min -40 °C and 240 min Annealing ramp 1 (°C / min) 0.5 °C / min Annealing temperature (°C) and retention time (h) Freezing ramp 2 (°C / min) -12 °C and 5 h 0.5 °C / min 0.5 °C / min Petition 870250078518, dated 03 / 09 / 2025, pp. 114 / 222 96 / 188 Freeze-drying Stage Parameter Cycle without standard CIN “Cycle without standard CIN” and without annealing Cycle CIN (center) Cycle CIN (low) Cycle CIN (high) Freezing temperature -45 °C -45 °C and (°C) / temp and 2 h 2 h retention time (h) PD ramp (°C / min) 0.3 °C / min 0.3 °C / min 0.3 °C / min 0.3 °C / min 0.3 °C / min Temperature -15 -8 Primary Drying (PD) PD (°C) and minimum retention time -25 °C and 26 h -25 °C and 27 h -8 °C and 13 or 50 h °C and 13 or 50 h °C and 13 or 50 h (h) PD pressure (mtorr) 70 mtorr 70 mtorr 100 mtorr 75 mtorr 100 mtor r Secondary Drying (SD) SD Ramp (°C / min) 0.1 °C / min 0.1 °C / min 0.1 °C / min 0.1 °C / min 0.1 °C / m in Petition 870250078518, dated 03 / 09 / 2025, pp. 115 / 222 97 / 188 Freeze-drying Stage Parameter Cycle without standard CIN Cycle without standard CIN and without annealing CIN cycle (center) CIN cycle (low) CIN cycle (high) SD temperature (°C) and retention time (h) SD pressure (mtorr) 30 °C and 8 h 70 mtorr 30 °C and 8 h 70 mtorr 40 °C and 8 h 100 mtorr 37 °C and 15 h 100 mtorr 45 °C and 8 h 100 mtorr Analysis of freeze-drying data

[0166] The freeze-drying process data were exported from the instrument and analyzed for primary drying time and total cycle time, as well as product temperatures. Primary drying was considered to have started at the beginning of the primary drying temperature ramp. The endpoint of the primary drying time was determined by observing when the instantaneous slope of the Pirani meter approached zero. The total freeze-drying time was the sum of the freeze-drying, primary drying, and secondary drying stages of the freeze-drying cycle.

[0167] The maximum product temperature of each flask containing a thermocouple was determined as the maximum temperature observed while the thermocouple remained inside the Petition 870250078518, dated 03 / 09 / 2025, pages 116 / 222 98 / 188 matrix frozen during primary drying. This was done for each thermocouple to obtain a range of product temperatures in the freeze-drying tray. Analytical assays - bispecific molecule BCMAxCD3 and bispecific molecule DLL3xCD3

[0168] Freeze-dried samples of bispecific molecules BCMAxCD3 and DLL3xCD3 were analyzed for product quality over 13 weeks. Samples were analyzed immediately after freeze-drying to the initial time point (time zero). The remaining samples were then randomly sorted (relative to the position in the freeze-drying tray) and placed at 4 °C, 25 °C and 40% relative humidity (25 °C / 40RH) and 40 °C and 65% relative humidity (40 °C / 65RH). Samples were retrieved from storage conditions for analytical testing at the specified time points (Table 5 and Table 7). Petition 870250078518, dated 03 / 09 / 2025, pages 117 / 222 99 / 188 Table 5. Information on Specifications of the bispecific molecule BCMAxCD3* Assay Specification Limits Time Points (wk) Cation Exchange HPLC (CEX-HPLC) Main Peak: 69.8% to 89.8% Acid Peaks: < 30.2% Basic Peaks: < 30.2% 0, 2, 4, 13 Size Exclusion Chromatography (SE-UHPLC) HMW: < 10.0% 0, 13 pH determination 4.0 to 4.4 0 Osmolality 250 to 350 mOsm / kg 0 Subvisible Particle Quantification HIAC < 6000 / 10 μm vial or more < 600 / 25 μm vial or more 0, 13 Moisture Content < 3.0% 0, 13 Bispecific molecule BCMAxCD3 Bispecific molecule BCMAxCD3 Bispecific molecule DLL3xCD3 Results and discussion Freeze-drying results: cycle time and maximum temperatures observed.

[0169] All freeze-drying cycles were successfully completed. The chamber pressure (measured by the capacitance manometer and the Pirani gauge), the shelf temperature, and the product temperatures for each freeze-drying cycle are given in Figure 2. Petition 870250078518, dated 03 / 09 / 2025, pp. 118 / 222 100 / 188

[0170] The freeze-drying cycle without CIN (Figure 2A) had a maximum product temperature of the bispecific molecule BCMAxCD3 of -33.3 °C and a maximum product temperature of the bispecific molecule DLL3xCD3 of -33.1 °C during primary drying. The freeze-drying cycle without CIN and without annealing (Figure 2B) had product temperatures of the bispecific molecule BCMAxCD3 2 °C higher (-31.3 °C) and product temperatures of the bispecific molecule DLL3xCD3 1.6 °C higher (-31.5 °C) than the freeze-drying cycle without CIN during primary drying at the same shelf temperature setpoint. This is consistent with the increased resistance to sublimation due to the removal of annealing. However, the freeze-drying cycle without CIN and without annealing had a similar primary drying endpoint when compared to the freeze-drying cycle without CIN with annealing.This indicates that annealing did not provide a time-saving benefit for these high-concentration bispecific molecules using the lyophilization cycle without CIN.

[0171] The CIN cycle (Figure 2C) with a primary drying shelf setpoint of -8 °C had a 50% reduction in primary drying time compared to the freeze-drying cycle. CIN can produce faster primary drying times because it completely removes stochastic freezing to form larger, more homogeneous ice crystals, which promote faster sublimation through a more porous cake structure.2 However, the observed maximum product temperatures for the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3 (27.8 °C and -28.7 °C, respectively) were also higher than Petition 870250078518, dated 03 / 09 / 2025, pages 119 / 222 101 / 188 that the previously reported critical temperature of -30 °C for the bispecific molecule BCMAxCD3 at 1 mg / mL and the bispecific molecule DLL3xCD3 at 5 mg / mL when using lyophilization with CIN.

[0172] A list of the minimum primary drying time and minimum total cycle time for the freeze-drying cycles tested is provided in Table 6. Since the primary drying time was determined when the instantaneous slope of the Pirani meter approached zero, it is considered the minimum primary drying time without the additional safety buffer that would be included in a typical freeze-drying recipe. The total cycle time calculated using the minimum primary drying time is therefore considered the minimum total cycle time. The freeze-drying cycle without CIN with annealing has a longer minimum total cycle time than the standard CIN-free cycle without annealing. This is because the two cycles had similar primary and secondary drying times, but the annealing step added 7 hours to the freeze-drying time of the standard cycle.

[0173] The CIN cycle had a primary drying endpoint of 13 hours, representing a 32% reduction in the minimum total cycle time. While this may change based on optimizing the primary drying shelf temperature to reduce maximum product temperatures, CIN has the potential to produce faster primary drying times, forming larger and more homogeneous ice crystals for a more porous cake structure. This, along with less time in the freeze phase, makes CIN the best option in terms of total cycle time. Petition 870250078518, dated 03 / 09 / 2025, pp. 120 / 222 102 / 188 Table 6. Primary drying time on a laboratory scale and total cycle time. Freeze-drying Process Laboratory Scale Minimum Time Minimum time to the final cycle point of total (h) primary drying (h) % Reduction in total cycle time compared to the freeze-drying cycle Freeze-drying cycle (annealing) without CIN 26 57 Freeze-drying cycle without CIN and without annealing 27 48 16 % CIN cycle 13 39 32 % HMWS Product Quality Results

[0174] After lyophilization, the samples were reconstituted and analyzed for % HMWS by SE-UHPLC, and the results were compared with the pre-lyophilized sample. The difference between the % HMWS of the reconstituted sample and the % HMWS of the sample before lyophilization was defined as A%HMWS. Although all results were within the specification limit of <10.0% HMWS, the A%HMWS after lyophilization was higher in the standard cycle samples with annealing for the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3. The standard cycle samples Petition 870250078518, dated 03 / 09 / 2025, pp. 121 / 222 103 / 188 without annealing showed the lowest HMWS growth after lyophilization.

[0175] The lyophilized CIN samples showed slightly more A%HMWS than those from the standard cycle without annealing, but still showed approximately 80% less A%HMWS than the samples from the standard cycle with annealing for the bispecific molecules BCMAxCD3 and DLL3xCD3. Because CIN samples spend less time in a partially frozen matrix, there is less risk of aggregation and HMWS formation in CIN lyophilization cycles than in annealing cycles.

[0176] After storing the lyophilized samples at 4 °C, 25 °C / 40RH and 40 °C / 65RH, the samples were removed at specified time points for % HMWS analysis. Samples of the bispecific molecule DLL3xCD3 were measured at time zero, 2 weeks and 4 weeks, and samples of the bispecific molecule BCMAxCD3 were measured at time zero and 13 weeks. Although the samples of the bispecific molecule BCMAxCD3 were originally planned to also have time points of 2 and 4 weeks, problems with the instrument resulted in a time point of 13 weeks being measured.

[0177] None of the freeze-drying groups showed an increase in % HMWS over time (%HMWS was not used in the case of comparison of stability time points). All samples of bispecific molecules BCMAxCD3 (Figure 4A) and DLL3xCD3 (Figure 4B) maintained % HMWS over time. The freeze-drying cycle with annealing consistently showed a higher % HMWS than the freeze-drying cycle without annealing and the CIN cycle. Petition 870250078518, dated 03 / 09 / 2025, pp. 122 / 222 104 / 188 Moisture content of the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3

[0178] All moisture results were < 3.0%. Three samples were measured at each time point, and samples from the front, center, and back of the freeze-drying shelf were measured at time zero, and no significant difference in moisture content was found between the samples from the three freeze-drying conditions (p > 0.05), as shown in Figure 5.

[0179] The CIN cycle in this example used the same primary drying temperature setpoint as the CIN cycle for the bispecific molecule BCMAxCD3 1 mg 6R. This resulted in a higher product temperature during primary drying of high-concentration bispecific molecules in this study than was observed in the previous study for the 1 mg / mL product due to the higher cake resistance in the high-concentration formulation. As discussed above, the product temperature of the CIN samples exceeded the previously characterized critical temperature of 30 °C for CIN-lyophilized bispecific molecules. It was observed in a previous study using standard 1 mg / mL and 5 mg / mL BiTE products that exceeding this temperature during primary drying caused an increase in moisture content over time, which was also observed in this example. Optimizing the primary drying temperature of the CIN lyophilization cycle would likely mitigate this problem. CEX results for the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3

[0180] The results of the CEX-HPLC assays of the bispecific molecules BCMAxCD3 and DLL3xCD3 showed differences Petition 870250078518, dated 03 / 09 / 2025, pages 123 / 222 The minimum values ​​for each lyophilization cycle were 105 / 188. A slight decrease in the main peak area and a corresponding slight increase in the acid and basic peak areas could be observed in samples of the bispecific molecule BCMAxCD3 stored at 40 °C / 65RH for 13 weeks, but this was observed independently of the lyophilization cycle (Figure 6A-C). The same behavior was observed for samples of the bispecific molecule DLL3xCD3 at 4 weeks (Figure 6D-F). Therefore, no difference was observed between the lyophilization cycle with and without annealing and the CIN cycle. Results of the quantification of HIAC particles of the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3. Quantification of particles of the bispecific molecule BCMAxCD3 (Table 7) and the bispecific molecule DLL3xCD3 (

[0181] Table 8) showed similar quantities of particles between samples from the lyophilization cycle with and without annealing and samples from the CIN cycle.All particle counts were well below the specification limit of 6000 particles / bottle > 10 μm and 600 particles / bottle > 25 μm. Table 7. Quantification of HIAC particles of the bispecific molecule BCMAxCD3 > 10 μm Standard Cycle (particles / bottle co) Standard Cycle without annealing (particles / bottle co) CIN Cycle (particles / bottle co) Time point (wk) 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH Petition 870250078518, dated 03 / 09 / 2025, pages 124 / 222 106 / 188 0 64 20 16 13 3 7 6 38 25 13 6 6 13 > 25 μm Standard Cycle (particles / bottle co) Standard Cycle without annealing (particles / bottle co) CIN Cycle (particles / bottle co) Time point (wk) 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 0 3 6 0 13 0 0 0 0 0 6 0 0 0 Table 8. Quantification of HIAC particles of the bispecific molecule DLL3xCD3 > 10 μm Standard Cycle (particles / bottle co) Standard Cycle without annealing (particles / bottle co) CIN Cycle (particles / bottle co) Time point (wk) 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 0 13 19 13 13 12 7 3 16 13 7 25 12 12 > 25 μm Standard Cycle (particles / bottle co) Standard Cycle without annealing CIN Cycle (particles / bottle co) Petition 870250078518, dated 03 / 09 / 2025, pages 125 / 222 107 / 188 (particles / bottle) Time point (wk) 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 40RH 40 °C / 65RH 4 °C 25 °C / 4 0RH 40 °C / 65RH 0 3 0 0 13 0 0 0 0 0 0 0 0 0 pH and Osmolality results for the bispecific molecule BCMAxCD3 and the bispecific molecule DLL3xCD3

[0182] At time zero, pH and osmolality measurements were performed on reconstituted samples from the standard cycle with and without annealing and from the CIN cycle. Osmolality and pH were tested to confirm the formulation and reconstitution of the product. No differences in pH or osmolality were observed between pre-lyophilization samples and reconstituted post-lyophilization samples for the bispecific molecule BCMAxCD3 (Table 9) and the bispecific molecule DLL3xCD3 (Table 10). Due to this, pH and osmolality were not monitored at any other time during the stability test. Table 9. pH and osmolality measurements of the bispecific molecule BCMAxCD3 Before lyophilization: Pre-lyophilization pH, Pre-lyophilization osmolality, DS of the bispecific molecule BCMAxCD3 4.28 319 After lyophilization Petition 870250078518, dated 03 / 09 / 2025, pages 126 / 222 108 / 188 Cycle Name pH at time zero Osmolality at time zero Standard Cycle 4.26 317 Standard cycle without annealing 4.25 315 CIN Cycle 4.26 318 Table 10. pH and osmolality measurements of the bispecific molecule DLL3xCD3 Before lyophilization Pre-lyophilization pH Pre-lyophilization osmolality DS of bispecific molecule DLL3xCD3 4.37 333 After lyophilization Cycle name pH at time zero Osmolality at time zero Standard cycle 4.35 333 Standard cycle without annealing 4.30 331 CIN cycle 4.30 333 Conclusions

[0183] Controlled ice nucleation lyophilization was evaluated in use with high-concentration products, using the bispecific molecule BCMAxCD3 (20 mg / mL) and the bispecific molecule DLL3xCD3 (15 mg / mL) in a 1.3 mL vial filled in 6R presentation as model proteins. CIN lyophilization was able to avoid the high HMWS levels commonly observed when using a CIN-free lyophilization cycle with annealing for these products. The samples Petition 870250078518, dated 03 / 09 / 2025, pp. 127 / 222 109 / 188 freeze-dried samples processed by CIN also showed less HMWS than freeze-dried and annealed samples over 13 weeks of product quality testing, regardless of storage conditions.

[0184] CIN freeze-drying also reduced the minimum total cycle time by 32% compared to the non-CIN freeze-drying cycle of bispecific molecules. Optimization of primary drying parameters leads to a smaller reduction in the minimum total cycle time. However, CIN freeze-drying primarily offers the benefit of reducing HMWS formation in high-concentration bispecific molecules, with the potential benefit of reducing the freeze-drying cycle time, making it an attractive option compared to a non-CIN freeze-drying cycle with annealing.

[0185] All references cited in this document, including patents, patent applications, literature publications and the like, are incorporated herein in their entirety by reference.

[0186] Although the present invention has been described with emphasis on preferred embodiments, it will be obvious to persons of ordinary skill in the art that variations of the preferred compounds and methods may be used and that it is intended that the invention may be practiced in a manner other than that specifically described herein. Consequently, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims. Table 13. Sequence table 1. CDR1 of artificial CD19 VL aa KASQSVDYDGDSYLN Petition 870250078518, dated 03 / 09 / 2025, pp. 128 / 222 110 / 188 2 . CD19 VL CDR2 artifi cial aa DASNLVS 3. CD19 VL CDR3 artifi cial aa QQSTEDPWT 4 . CDR1 and VH of CD19 artificial aa SYWMN 5 . CD19 VH CDR2 artifi cial aa QIWPGDGDTNYNGKFKG 6. CD19 VH CDR3 artifi cial aa RETTTVGRYYYAMDY 7 . VL de CD19 artifi cial aa DIQLTQSPASLAVSLGQRATISCKASQSV DYDGDSYLNWYQQIPGQPPKLLIYDASNL VSGIPPRFSGSGSGTDFTLNIHPVEKVDA ATYHCQQSTEDPWTFGGGTKLEIK 8. VH de CD19 artifi cial QVQLQS LQS LQS GSSVKISCKASGYAF SSYWMNWVKQRPGQGLEWIGQIWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLA SEDSAVYFCARRETTTVGRYYYAMDYWGQ GTTVTVSS 9. CD3 VH CDR1 artifi cial aa RYTMH 10 VH CDR1 artifi cial aa RYTMH 10 VH CDR2 artifi CDRa. YINPSRGYTNYNQKFKD 11. CD3 VH CDR3 artifi cial aa YYDDHYCLDY 12 . CD3 VL CDR1 artifi cial aa RASSSVSYMN 13. CD3 VL CDR2 artifi cial aa DTSKVAS Petition 870250078518, of 03 / 09 / 2025, p. 129 / 222 111 / 188 14. CD3 VL CDR3 artifi cial aa QQWSSNPLT 15. CD3 VH artifi cial aa DIKLQQSGAELARPGASVKMSCKTSGYTF TRYTMHWVKQRPGQGL EWIGYINPSRGYTNYNQKFKDKATLTTDK SSSTAYMQLSSLTSEDSAVYYCARYYDDH YCLDYWGQGTTLTVSS 16. CD3 VL artifi cial aa VDDIQLTQSPAIMSASPGEKVTMTCRASS SVSYMNWYQQKSGTSPKRWIYDTSKVASG VPYRFSGSGSGTSYSLTISSMEAEDAATY YCQQWSSNPLTFGAGTKLELK 17. scFv CD19xCD3, incl ligador e marcador de his artifi cial aa DIQLTQSPASLAVSLGQRATISCKASQSV DYDGDSYLNWYQQIPGQPPKLLIYDASNL VSGIPPRFSGSGSGTDFTLNIHPVEKVDA ATYHCQQSTEDPWTFGGGTKLEIKGGGGS GGGGSGGGGSQVQLQQSGAELVRPGSSVK ISCKASGYAFSSYWMNWVKWIQRPGQGLE GQIWP GD GD TNYNGKFKGKATLTADES SS TAYMQLSSLASEDSAVYFCARRETTTVGR YYYAMDYWGQGTTVTVSSGGGGSDIKLQQ SGAELARPGASVKMSCKTSGYTFTRYTMH WVKQRPGQGLEWIGYINPSRGYTNYNQKF KDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEG GSGGSGGSGGSGGVDDIQLTQSPAIMSAS PGEKVTMTCRASSSVSYMNWYQQKSGTSP KRWIYDTSKVASGVPYRFSGSGTSSYSL TISSMEAEDAATYYCQQWSSNPLTFGAGT KLELKHHHHHH Petition: 870250078518, on September 3, 2025, page. 130 / 222 112 / 188 18. CDR-L1 of artificial I2C aa GSSTGAVTSGNYPN 19. CDR-L2 of artificial I2C aa GTKFLAP 20. CDR-L3 of artificial I2C aa VLWYSNRWV 21. CDR-H1 of artificial I2C aa 22 KYAMN . CDR-H2 de I2C artifi cial aa RIRSKYNNYATYYADSVKD 23. CDR-H3 de I2C artifi cial aa HGNFGNSYISYWAY 24. VH de I2C artifi cial aa EVQLVESGGGLVQPGGSLKLSCAASGFTF NKAPYARGWRGWWNW ATYYAD SVKD RFTISRDDS KNTAYLQMNN LKTEDTAVYYCVRHGNFGNSYISYISYWAYWG QGTLVTVSS 25. VL de I2C artifi cial aa QTVVTQEPSLTVSPGGTVTLTCGSSTGAV TSGNYPNWVQQKPGQQAPRGGTKFLG GTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWVFGGGTKLTVL 26. VH-VL de I2C artifi cial aa EVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAMNWVRQAPGKGLEWVARIRSKYD NYD SVRDKYD KNTAYLQMNN LKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVSSGGGGSGGGGGGGSQTVVT QEPSLTVSPGGTVTLTCGSSTGAVTSGNY PNWVQQKPGQAPRGLIGGTKFLAPGTPAR FSGSLLGGKALSGTVYCVYC WYSNRWVFGGGTKLTVL Petition 870250078518, of 03 / 09 / 2025, p. 131 / 222 113 / 188 27. CD33 ccVH de E11 artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQCLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSS 28. CD33 VH de E11 Artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQGLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSS 29. CD33 HCDR1 de E11 artifi cial aa NYGMN 30. CD33 HCDR2 de E11 artifi cial aa WINTYTGEPTYADKFQG 31. CD33 HCDR3 de E11 artifi cial aa WSWSDGYYVYFDY 32. CD33 CC VL de E11 artifi cial aa DIVMTQSPDSLTVSLGERTTINCKSSQSV LDSSTNKNSLAWYQQKPGQPPKLLLSWAS TRESGIPDRFSGSGSGTDFTLTIDSPQPE DSATYYCQQSAHFPITFGCGTRLEIK 33. CD33 VL de E11 artifi cial aa DIVMTQSPDSLTVSLGERTTINCKSSQSV LDSSTNKNSLAWYQQKPGQPPKLLLSWAS TRESGIPDRFSGSGSGTDFTLTIDSPQPE DSATYYCQQSAHFPITFGQGTRLEIK 34. CD33 LCDR1 from E11 artificial aa KSSQSVLDSSTNKNSLA Petition: 870250078518, on September 3, 2025, page. 132 / 222 114 / 188 35. CD33 LCDR2 de E11 artifi cial aa WASTRES 36. CD33 LCDR3 de E11 artifi cial aa QQSAHFPIT 37. CD33 HL CC de E11 artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQCLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLTVSLGERTTINCKSSQSVLDSSTNKN SLAWYQQKPGQPPKLLLSWASTRESGIPD RFSGSGSGTDFTLTIDSPQPEDSATYYCQ QSAHFPITFGCGTRLEIK 38. CD33 HL of E11 artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQGLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLTVSLGERTTINCKSSQSVLDSSTNKN SLAWYQQKPGQPPKLLLSWASTRESGIPD RFSGSGSGTDFTLTIDSPQPEDSATYYCQ QSAHFPITFGQGTRLEIK 39. Molécula biespecíf ica CD33 CC E11 HL x I2C HL artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQCLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLTVSLGERTTINCKSSQSVLDSSTNKN Petition: 870250078518, on September 3, 2025, page. 133 / 222 115 / 188 SLAWYQQKPGQPPKLLLSWASTRESGIPD RFSGSGSGTDFTLTIDSPQPEDSATYYCQ QSAHFPITFGCGTRLEIKSGGGGSEVQLV ESGGGLVQPGGSLKLSCAASGFTFNKYAM NWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTED TAVYYCVRHGNFGNSYISYWAYWGQGTLV TVSSGGGGSGGGGSGGGGSQTVVTQEPSL TVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNR WVFGGGTKLTVL 40. CD33 E11 HL x I2C HL artifi cial aa MGWSCIILF LVATAT GVHS QVQ LVQ S GAE VKKPGESVKVSCKASGYTFTNYGMNWVKQ APGQGLEWMGWINTYTGEPTYADKFQGRV TMTTDTSTSTAYMEIRNLGGDDTAVYYCA RWSWSDGYYVYFDYWGQGTSVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLTVSLGER TTINCKSSQSVLDSSTNKNSLAWYQQKPG QPPKLLLSWASTRESGIPDRFSGSGSGTD FTLTIDSPQPEDSATYYCQQSAHFPITFG QGTRLEIKSGGGGSEVQLVESGGGLVQPG GSLKLS CAAS GFTFNKYAMNWVRQAP GKG LEWVARIRSKYNNYATYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHG NFGNSYISYWAYWGQGTLVTVSSGGGGSG GGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGNYPNWVQQKPGQAPRGL IGGTKFLAPGTPARFSGSLLGGKAALTLS Petição 870250078518, de 03 / 09 / 2025, pág. 134 / 222 116 / 188 GVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLHHHHHH 41. Molécula HLE biespecíf ica CD33 CC x I2C- scFc artifi cial aa QVQLVQSGAEVKKPGESVKVSCKASGYTF TNYGMNWVKQAPGQCLEWMGWINTYTGEP TYADKFQGRVTMTTDTSTSTAYMEIRNLG GDDTAVYYCARWSWSDGYYVYFDYWGQGT SVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLTVSLGERTTINCKSSQSVLDSSTNKN SLAWYQQKPGQPPKLLLSWASTRESGIPD RFSGSGSGTDFTLTIDSPQPEDSATYYCQ QSAHFPITFGCGTRLEIKSGGGGSEVQLV ESGGGLVQPGGSLKLSCAASGFTFNKYAM NWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTED TAVYYCVRHGNFGNSYISYWAYWGQGTLV TVSSGGGGSGGGGSGGGGSQTVVTQEPSL TVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNR WVFGGGTKLTVLGGGGDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKP CEEQYGSTYRCVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDV Petition 870250078518, dated 03 / 09 / 2025, pages 135 / 222 117 / 188 SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 42 . EGFRvIIIx CD3-scFc VH CDR1 artificial aa nygmh 43. EGFRvIIIx CD3-scFc VH CDR2 artificial aa viwydgsdkyyadsvrg 44 . EGFRvIIIx CD3-scFc VH CDR3 artificial aa dgydiltgnprdfdy 45. EGFRvIIIx CD3-scFc VL CDR1 artificial aa rssqslvhsdgntyls 46. EGFRvIIIx CD3-scFc VL CDR2 artificial aa risrrfs 47 . EGFRvIIIx CD3-scFc VL CDR3 artificial aa mqsthvprt 48. EGFRvIII CCxCD3scFc VH artificial aa qvqlvesgggvvqsgrslrlscaasgftf rnygmhwvrqapgkclewvaviwydgsdk yyadsvrgrftisrdnskntlylqmnslr aedtavyycardgydiltgnprdfdywgq gtlvtvss Petition 870250078518, dated 03 / 09 / 2025, pages 136 / 222 118 / 188 49. EGFRvIII CCxCD3scFc VL artifi cial aa dtvmtqtplsshvtlgqpasiscrssqsl vhsdgntylswlqqrpgqpprlliyrisr rfsgvpdrfsgsgagtdftleisrveaed vgvyycmqsthvprtfgcgtkveik 50. EGFRvIII CCxCD3scFc scFv artifi cial aa qvqlvesgggvvqsgrslrlscaasgftf rnygmhwvrqapgkclewvaviwydgsdk yyadsvrgrftisrdnskntlylqmnslr aedtavyycardgydiltgnprdfdywgq gtlvtvssggggsggggsggggsdtvmtq tplsshvtlgqpasiscrssqslvhsdgn tylswlqqrpgqpprlliyrisrrfsgvp drfsgsgagtdftleisrveaedvgvyyc mqsthvprtfgcgtkveik 51.Molécula biespecíf ica EGFRvIII CCxCD3scFc artifi cial aa qvqlvesgggvvqsgrslrlscaasgftf rnygmhwvrqapgkclewvaviwydgsdk yyadsvrgrftisrdnskntlylqmnslr aedtavyycardgydiltgnprdfdywgq gtlvtvssggggsggggsggggsdtvmtq tplsshvtlgqpasiscrssqslvhsdgn tylswlqqrpgqpprlliyrisrrfsgvp drfsgsgagtdftleisrveaedvgvyyc mqsthvprtfgcgtkveiksggggsevql vesggglvqpggslklscaasgftfnkya mnwvrqapgkglewvarirskynnyatyy adsvkdrftisrddskntaylqmnnlkte dt avyycvrhgn f gnsyisywaywgqgtl vtvssggggsggggsggggsqtvvtqeps ltvspggtvtltcgsstgavtsgnypnwv qqkpgqaprgliggtkflapgtparfsgs. Petição 870250078518, de 03 / 09 / 2025, pág. 137 / 222 119 / 188 llggkaaltlsgvqpedeaeyycvlwysn rwvfgggtkltvl 52 . Molécula HLE biespecíf ica EGFRvIII CCxCD3scFc artifi cial aa qvqlvesgggvvqsgrslrlscaasgftf rnygmhwvrqapgkclewvaviwydgsdk yyadsvrgrftisrdnskntlylqmnslr aedtavyycardgydiltgnprdfdywgq gtlvtvssggggsggggsggggsdtvmtq tplsshvtlgqpasiscrssqslvhsdgn tylswlqqrpgqpprlliyrisrrfsgvp drfsgsgagtdftleisrveaedvgvyyc mqsthvprtfgcgtkveiksggggsevql vesggglvqpggslklscaasgftfnkya mnwvrqapgkglewvarirskynnyatyy adsvkdrftisrddskntaylqmnnlkte dtavyycvrhgn fgnsyisywaywgqgtl vtvssggggsggggsggggsqtvvtqeps ltvspggtvtltcgsstgavtsgnypnwv qqkpgqaprgliggtkflapgtparfsgs llggkaaltlsgvqpedeaeyycvlwysn rwvfgggtkltvlggggdkthtcppcpap ellggpsvflfppkpkdtlmisrtpevtc vvvdvshedpevkfnwyvdgvevhnaktk pceeqygstyrcvsvltvlhqdwlngkey kckvsnkalpapiektiskakgqprepqv ytlppsreemtknqvsltclvkgfypsdi avewesngqpennykttppvldsdgsffl yskltvdksrwqqgnvfscsvmhealhnh ytqkslslspgkggggsggggsggggsgg ggsggggsggggsdkthtcppcpapellg gpsvflfppkpkdtlmi s rtpevt cvvvd Petition: 870250078518, on September 3, 2025, page. 138 / 222 120 / 188 vshedpevkfnwyvdgvevhnaktkpcee qygstyrcvsvltvlhqdwlngkeykckv snkalpapiektiskakgqprepqvytlp psreemtknqvsltclvkgfypsdiavew esngqpennykttppvldsdgsfflyskl tvdksrwqqgnvfscsvmhealhnhytqk slslspgk 53. MSLN_5 VH CDR1 artifi cial aa DYYMT 54. MSLN_5 VH CDR2 artifi cial aa YISSSGSTIYYADSVKG 55. MSLN_5 VH CDR3 artifi cial aa DRNSHFDY 56. MSLN_5 VL CDR1 artifi cial aa RASQGINTWLA 57. MSLN_5 VL CDR2 artificial aa GASGLQS 58. MSLN_5 VL CDR3 artifi cial aa QQAKSFPRT 59. MSLN_5 VH artifi cial aa QVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQAPGKGLEWLSYISSSGSTI YYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDRNSHFDYWGQGTLVTVS S 60. MSLN_5 VL artifi cial aa DIQMTQSPSSVSASVGDRVTITCRASQGI NTWLAWYQQKPGKAPKLLIYGASGLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKSFPRTFGQGTKVEIK 61. MSLN_5 scFv artifi cial aa QVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQAPGKGLEWLSYISSSGSTI Petition: 870250078518, on September 3, 2025, page. 139 / 222 121 / 188 YYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDRNSHFDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSVSA SVGDRVTITCRASQGINTWLAWYQQKPGK APKLLIYGASGLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQAKSFPRTFGQ GTKVEIK 62. MSLN_5xI2 C0 molécula biespecíf ica artifi cial aa QVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQAPGKGLEWLSYISSSGSTI YYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDRNSHFDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSVSA SVGDRVTITCRASQGINTWLAWYQQKPGK APKLLIYGASGLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQAKSFPRTFGQ GTKVEIKSGGGGSEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLT CGSSTGAVTSGNYPNWVQQKPGQAPRGLI GGTKFLAPGTPARFSGSLLGGKAALTLSG VQPEDEAEYYCVLWYSNRWVFGGGTKLTV L 63. Molécula HLE biespecíf ica artifi cial aa QVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQAPGKGLEWLSYISSSGSTI YYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDRNSHFDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSVSA Petition 870250078518, de 03 / 09 / 2025, pág. 140 / 222 122 / 188 MSLN_5xCD 3-scFc SVGDRVTITCRASQGINTWLAWYQQKPGK APKLLIYGASGLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQAKSFPRTFGQ GTKVEIKSGGGGSEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGGSGG GGSGGGSQTVVTQEPSLTVSPGGTVTLT CGSSTGAVTSGNYPNWVQQKPGQAPRGLI GGTKFLAPGTPARFSGSLLGGKAALTLSG VQPEDEAEYYCVLWYSNRWVFGGGTKLTV LGGGDGDKTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSGGGSGGGGSGGGG SDKTHTCPPPAPELLGGPSVFLFPPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPCEEEQYGSTYRCVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGPYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLPGK Petition 870250078518, dated 03 / 09 / 2025, pp. 141 / 222 123 / 188 64. Molécula HLE biespecíf ica MSLN_5_CC xCD3-scFc artifi cial aa QVQLVESGGGLVKPGGSLRLSCAASGFTF SDHYMSWIRQAPGKCLEWFSYISSSGGII YYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARDVGSHFDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSVSA SVGDRVTITCRASQDISRWLAWYQQKPGK APKLLISAASRLQSGVPSRFSGSGSGTDF TLTISSLQPEDFAIYYCQQAKSFPRTFGC GTKVEIKSGGGGSEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLT CGSSTGAVTSGNYPNWVQQKPGQAPRGLI GGTKFLAPGTPARFSGSLLGGKAALTLSG VQPEDEAEYYCVLWYSNRWVFGGGTKLTV LGGGGDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSGGGGSGGGGSGGGG SDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPCEEQYGSTYRCVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKT Petition: 870250078518, on September 3, 2025, page. 142 / 222 124 / 188 ISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 65. CDR-H1 de CDH19 65254.007 artifi cial aa SYGMH 66. CDR-H2 de CDH19 65254.007 artifi cial aa FIWYEGSNKYYAESVKD 67. CDR-H3 de CDH19 65254.007 artifi cial aa RAGIIGTIGYYYGMDV 68. CDR-L1 de CDH19 65254.007 artifi cial aa SGDRLGEKYTS 69. CDR-L2 de CDH19 65254.007 artifi cial aa QDTKRPS 70. CDR-L3 de CDH19 65254.007 artifi cial aa QAWESSTVV 71. VH de CDH19 65254.007 artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKGLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSS 72. VL de CDH19 65254.007 artifi cial aa SYELTQPPSVSVSPGQTASITCSGDRLGE KYTSWYQQRPGQSPLLVIYQDTKRPSGIP Petition: 870250078518, on September 3, 2025, page. 143 / 222 125 / 188 ERFSGSNSGNTATLTISGTQAMDEADYYC QAWESSTVVFGGGTKLTVLS 73. VH-VL de CDH19 65254.007 artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKGLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGGGTKLTVLS 74 . CDH19 65254.007 QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGGGTKLTVLSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLHHHHHH Petition: 870250078518, on September 3, 2025, page. 144 / 222 126 / 188 75. Molécula HLE biespecíf ica CDH19 65254.007 x I2C — scFc artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF S S YGMHWVRQAP GKGLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGGGTKLTVLSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLGGGGDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGST YRCVSVLTVLHQDWLNGKEYKCKVSNKAL Petition 870250078518, dated 03 / 09 / 2025, pp. 145 / 222 127 / 188 PAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 76. Molecule HLE biespecíf ica CDH19 65254.007 x I2C scFc_delG K artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SS YGMHWVRQAP GKGLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGGGTKLTVLSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAV YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLGGGGDKTHTCPPPAPELLGG PSVFLFPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS Petition 870250078518, dated 03 / 09 / 2025, pp. 146 / 222 128 / 188 LSLSPGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPCEEQYGSTYR CVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K 77. CDH19 65254.007 CC x I2C -SCFc VH artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSS 78. CDH19 65254.007 CC x I2C -SCFc VL artifi cial aa SYELTQPPSVSVSPGQTASITCSGDRLGE KYTSWYQQRPGQSPLLVIYQDTKRPSGIP ERFSGSNSGNTATLTISGTQAMDEADYYC QAWESSTVVFGCGTKLTVL 79. CDH19 65254.007 CC x I2C -scFc SCFv artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGCGTKLTVL 80.Artificial bispecific molecule aa QVQLVESGGGVVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAFIWYEGSNK. Petition 870250078518, dated 03 / 09 / 2025, pp. 147 / 222 129 / 188 ica CDH19 65254.007 CC x I2C -scFc YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGCGTKLTVLSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVL 81. Molécula HLE biespecíf ica CDH19 65254.007 CC x I2C -scFc artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITCSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGCGTKLTVLSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSP Petition 870250078518, dated 03 / 09 / 2025, pp. 148 / 222 130 / 188 GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLGGGGDKTHTCPPCPAPELLGG PSVFLFPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGSGGGGSGG GGSGGGGSDKTHTCPPCPAPELLGGPSVF LFPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEEQYGST YRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 82 . Molecule HLE biespecíf ica CDH19 65254.007 CC x I2C scFc_delG K artifi cial aa QVQLVESGGGVVQPGGSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAFIWYEGSNK YYAESVKDRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARRAGIIGTIGYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSSYELT QPPSVSVSPGQTASITSGDRLGEKYTSW YQQRPGQSPLLVIYQDTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWES STVVFGCTKLTVLSGGGGSEVQLVESGG Petition 870250078518, de 03 / 09 / 2025, pág. 149 / 222 131 / 188 GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNF GNSYIS YWAYWGQGTLVTVS S GGGGSGGGGSGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLGGGGDKTHTCPPCPAPELLGG PSVFLFPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPCEEQYGSTYR CVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K 83. FLT3_7 A8xCD3scFc VH CDR1 artificial aa NARMGVS Petition 870250078518, de 03 / 09 / 2025, pág. 150 / 222 132 / 188 84. FLT3_7 A8xCD3scFc VH CDR2 artificial aa HIFSNDEKSYSTSLKN 85. FLT3_7 A8xCD3scFc VH CDR3 artificial aa IVGYGSGWYGFFDY 86. FLT3_7 A8xCD3scFc VL CDR1 artificial aa RASQGIRNDLG 87. FLT3_7 A8xCD3scFc VL CDR2 artificial aa AASTLQS 88. FLT3_7 A8xCD3scFc VL CDR3 artificial aa LQHNSYPLT 89. FLT3_7 A8xCD3scFc VH artificial aa QVTLKESGPTLVKPTETLTLTCTLSGFSL NNARMGVSWIRQPPGKCLEWLAHIFSNDE KSYSTSLKNRLTISKDSSKTQVVLTMTNV DPVDTATYYCARIVGYGSGWYGFFDYWGQ GTLVTVSS 90. FLT3_ A8scFc VL artifi cial aa DIQMTQSPSSLSASVGDRVTITCRASQGI RNDLGWYQQKP GKAP KRLIYAAS TLQSGV PSRFSGSGSGTEFTLTISSLQPEDFATYY CLQHNSYPLTFGCGTKVEIK Petition: 870250078518, on September 3, 2025, page. 151 / 222 133 / 188 91. FLT3_7 A8xCD3scFv artifi cial aa QVTLKESGPTLVKPTETLTLTCTLSGFSL NNARMGVSWIRQPPGKCLEWLAHIFSNDE KSYSTSLKNRLTISKDSSKTQVVLTMTNV DPVDTATYYCARIVGYGSGWYGFFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQGIRNDLGW YQQKPGKAPKRLIYAASTLQSGVPSRFSG SGSGTEFTLTISSLQPEDFATYYCLQHNS YPLTFGCGTKVEIK 92. Molécula biespecíf ica FLT3_7 A8xCD3 artifi cial aa QVTLKESGPTLVKPTETLTLTCTLSGFSL NNARMGVSWIRQPPGKCLEWLAHIFSNDE KSYSTSLKNRLTISKDSSKTQVVLTMTNV DPVDTATYYCARIVGYGSGWYGFFDYWGQ GTLVTVSSGGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQGIRNDLGW YQQKPGKAPKRLIYAASTLQSGVPSRFSG SGSGTEFTLTISSLQPEDFATYYCLQHNS YPLTFGCGTKVEIKSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVL 93. Molécula HLE biespecíf artifi cial aa QVTLKESGPTLVKPTETLTLTCTLSGFSL NNARMGVSWIRQPPGKCLEWLAHIFSNDE KSYSTSLKNRLTISKDSSKTQVVLTMTNV Petition 870250078518, dated 03 / 09 / 2025, pp. 152 / 222 134 / 188 ica FLT3_7 A8xCD3scFc DPVDTATYYCARIVGYGSGWYGFFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQGIRNDLGW YQQKPGKAPKRLIYAASTLQSGVPSRFSG SGSGTEFTLTISSLQPEDFATYYCLQHNS YPLTFGCGTKVEIKSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVR QAPGKGLEWVARIRSKYNNYATYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVLGGGGDKTHTCPPPAPELLGG PSVFLFPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPCEEQ YGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGSGGGSGG GGSGGGGSDKTHTCPPPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGST YRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKS Petition 870250078518, de 03 / 09 / 2025, pág. 153 / 222 135 / 188 RWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 94. VH CDR1 DLL3_1_CC delGK artifi cial aa SYYWS 95. VH CDR2 DLL3_1_CC delGK artifi cial aa YVYYSGTTNYNPSLKS 96. VH CDR3 DLL3_1_CC delGK artifi cial aa IAVTGFYFDY 97. VL CDR1 DLL3_1_CC delGK artifi cial aa RASQRVNNNYLA 98. VL CDR2 DLL3_1_CC delGK artifi cial aa GASSRAT 99. VL CDR3 DLL3_1_CC delGK artifi cial aa QQYDRSPLT 100. VH DLL3_1_CC delGK artifi cial aa QVQLQESGPGLVKPSETLSLTCTVSGGSI SSYYWSWIRQPPGKCLEWIGYVYYSGTTN YNPSLKSRVTISVDTSKNQFSLKLSSVTA ADTAVYYCASIAVTGFYFDYWGQGTLVTV SS 101. VL DLL3_1_CC delGK artifi cial aa EIVLTQSPGTLSLSPGERVTLSCRASQRV NNNYLAWYQQRPGQAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYDRSPLTFGCGTKLEIK Petition: 870250078518, on September 3, 2025, page. 154 / 222 136 / 188 102. DLL3_1_CC delGK artifi cial aa QVQLQESGPGLVKPSETLSLTCTVSGGSI SSYYWSWIRQPPGKCLEWIGYVYYSGTTN YNPSLKSRVTISVDTSKNQFSLKLSSVTA ADTAVYYCASIAVTGFYFDYWGQGTLVTV SSGGGGSGGGGSGGGGSEIVLTQSPGTLS LSPGERVTLSCRASQRVNNNYLAWYQQRP GQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYDRSPLTF GCGTKLEIK 103. Bispecific molecule DLL3_1_CC xCD3_delG K artifi cial aa QVQLQESGPGLVKPSETLSLTCTVSGGSI SSYYWSWIRQPPGKCLEWIGYVYYSGTTN YNPSLKSRVTISVDTSKNQFSLKLSSVTA ADTAVYYCASIAVTGFYFDYWGQGTLVTV SSGGGGSGGGGSEIVLTQSPGTLS LSPGERVTLSCRASQRVNNNYLAWYQQRP GQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYDRSPLTF GCGTKLEIKSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYCVRH GNFGNSYISYWAYWGQGTLVVSSGGGS GGGGSGGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVL 104. Molécula HLE biespíf artifi cial aa QVQLQESGPGLVKPSETLSLTCTVSGGSI SSYYWSWIRQPPGKCLEWIGYVYYSGTTN YNPSLKSRVTISVDTSKNQFSLKLSSVTA Petition 870250078518, de 03 / 09 / 2025, pág. 155 / 222 137 / 188 ica DLL3_1_CC xCD3scFc_delG K ADTAVYYCASIAVTGFYFDYWGQGTLVTV SSGGGGSGGGGSGGGGSEIVLTQSPGTLS LSPGERVTLSCRASQRVNNNYLAWYQQRP GQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYDRSPLTF GCGTKLEIKSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRH GNFGNSYISYWAYWGQGTLVTVSSGGGGS GGGGSGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVLGGGGDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPCEQYGSTY RCVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GGGGSGGGGSGGGSGGGGSGGGGSGGGG SDKTHTCPPPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPCEEQYGSTYRCVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKT Petition 870250078518, of 03 / 09 / 2025, p. 156 / 222 138 / 188 TPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 105. VH CDR1 CD19 97- G1RE-C2 artifi cial aa SYGMH 106. VH CDR2 CD19 97- G1RE-C2 artifi cial a VISGYNK. VH CDR3 CD19 97- G1RE-C2 artifi cial aa DRGTIFGNYGLEV 108. VH CD19 97-G1RE- C2 CC artifi cial aa QVQLVESGGVVQPGRSLRLSCAASGFTF SSYGMHWVRQAPGKCLEWVAVISNKYEGSYEGS YYAESVKGRFTISRDNSKNTLYLQMNSLR DEDTAVYYCARDRGTIFGNYGLEVWGQGT TVTVSS 109. VL CDR1 CD19 97- G1RE-C2 artifi cial aa RSSQSLLHKNAFNYLD 110. VL CDR2 CD19 97- C-a G1cial LGSREAS LGS 111. VL CDR3 CD19 97- G1RE-C2 artifi cial aa MQALQTPFT 112 . VL CD19 97-G1RE- C2 CC artifi cial aa DIVMTQSPLSLPVISGEPASISCRSSQSL LHKNAFNYLDWYLQKPGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQALQTPFTFGCGTKVDIK Petition 870250078518, of 03 / 09 / 2025, p. 157 / 222 139 / 188 113. CD19 97- G1RE-C2 CC x I2C0 artifi cial aa MDMRVPAQLLGLLLLWLRGARCDIVMTQS PLSLPVISGEPASISCRSSQSLLHKNAFN YLDWYLQKPGQSPQLLIYLGSNRASGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPFTFGCGTKVDIKGGGGSGGGGSG GGGSQVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMHWVRQAPGKCLEWVAVISYE GSNKYYAESVKGRFTISRDNSKNTLYLQM NSLRDEDTAVYYCARDRGTIFGNYGLEVW GQGTTVTVSSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRH GNFGNSYISYWAYWGQGTLVTVSSGGGGS GGGGSGGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVL 114 . CD19 97- G1RE-C2 CC x l2C0-scFc artifi cial aa MDMRVPAQLLGLLLLWLRGARCDIVMTQS PLSLPVISGEPASISCRSSQSLLHKNAFN YLDWYLQKPGQSPQLLIYLGSNRASGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPFTFGCGTKVDIKGGGGSGGGGSG GGGSQVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMHWVRQAPGKCLEWVAVISYE GSNKYYAESVKGRFTISRDNSKNTLYLQM NSLRDEDTAVYYCARDRGTIFGNYGLEVW GQGTTVTVSSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK Petition 870250078518, dated 03 / 09 / 2025, pages 158 / 222 140 / 188 GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRH GNFGNSYISYWAYWGQGTLVTVSSGGGGS GGGGSGGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVLGGGGDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPCEEQYGSTY RCVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 115. VH CDR1 CDH3 G8A 6-B12 artifi cial aa SYPIN 116. VH CDR2 CDH3 G8A 6-B12 artificial aa VIWTGGGTNYASSVKG Petition 870250078518, dated 03 / 09 / 2025, pages 159 / 222 141 / 188 117. VL CDR3 CDH3 G8A 6-B12 artificial aa SRGVYDFDGRGAMDY 118. CDH3 G8A 6-B12 artificial aa QQYYSYPYT 121. EDTAVYYCAKSRGVYDFDGRGAMDYWGQG TLVTVSS 122. VL CDH3 G8A 6-B12 artificial aa DIVMTQSPDSLAVSLGERATINCKSSQSL LYSSNQKNYFAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSYPYTFGQGTKLEIK 123. scFv CDH3 G8A 6-B12 artifi cial aa EVQLLESGGGLVQPGGSLRLSCAASGFSF SSYPINWVRQAPGKGLEWVGVIWTGGGTN YASSVKGRFTISRDNSKNTVYLQMNSLRA EDTAVYYCAKSRGVYDFDGRGAMDYWGQG TLVTVSSGGGGSGGGGSGGGGSDIVMTQS PDSLAVSLGERATINCKSSQSLLYSSNQK NYFAWYQQKPGQPPKLLIYWASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSYPYTFGQGTKLEIK Petition: 870250078518, on September 3, 2025, page. 160 / 222 142 / 188 124 . Molécula biespecíf ica CDH3 G8A 6-B12 x I2C0 artifi cial aa EVQLLESGGGLVQPGGSLRLSCAASGFSF SSYPINWVRQAPGKGLEWVGVIWTGGGTN YASSVKGRFTISRDNSKNTVYLQMNSLRA EDTAVYYCAKSRGVYDFDGRGAMDYWGQG TLVTVSSGGGGSGGGGSGGGGSDIVMTQS PDSLAVSLGERATINCKSSQSLLYSSNQK NYFAWYQQKPGQPPKLLIYWASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSYPYTFGQGTKLEIKSGGGGSEVQL VESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHGNFGNSYISYWAYWGQGTL VTVSSGGGGSGGGGSGGGGSQTVVTQEPS LTVSPGGTVTLTCGSSTGAVTSGNYPNWV QQKPGQAPRGLIGGTKFLAPGTPARFSGS LLGGKAALTLSGVQPEDEAEYYCVLWYSN RWVFGGGTKLTVL 125.Molécula HLE biespecíf ica CDH3 G8A 6-B12 x I2C0 artifi cial aa EVQLLESGGGLVQPGGSLRLSCAASGFSF SSYPINWVRQAPGKGLEWVGVIWTGGGTN YASSVKGRFTISRDNSKNTVYLQMNSLRA EDTAVYYCAKSRGVYDFDGRGAMDYWGQG TLVTVSSGGGGSGGGGSGGGGSDIVMTQS PDSLAVSLGERATINCKSSQSLLYSSNQK NYFAWYQQKPGQPPKLLIYWASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSYPYTFGQGTKLEIKSGGGGSEVQL VESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTE. Petition: 870250078518, on September 3, 2025, page. 161 / 222 143 / 188 DTAVYYCVRHGNFGNSYISYWAYWGQGTL VTVSSGGGGSGGGGSGGGGSQTVVTQEPS LTVSPGGTVTLTCGSSTGAVTSGNYPNWV QQKPGQAPRGLIGGTKFLAPGTPARFSGS LLGGKAALTLSGVQPEDEAEYYCVLWYSN RWVFGGGTKLTVLGGGGDKTHTCPPCPAP ELLGGPSVFLFPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVNHAKTK PCEEQYGSTYRCVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGGGGSDKTHTCPPPAPELLG GPSVFLFPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 126. BCMA A7 27-C4-G7 CDR1 VH artifi cial aa NHIIH 127 . BCMA A7 27-C4-G7 CDR2 VH artifi cial aa YINPYPGYHAYNEKFQG Petition 870250078518, de 03 / 09 / 2025, pág. 162 / 222 144 / 188 128. BCMA A7 27-C4-G7 CDR3 VH artificial cial aa DGYYRDTDVLDY 129. BCMA A7 27-C4-G7 CDR1 VL artificial cial aa QASQDISNYLN 130. BCMA A7 27-C4-G7 CDR2 VL artificial cial aa YTSRLHT 131. BCMA A7 27-C4-G7 CDR3 VL artificial cial aa QQGNTLPWT 132. BCMA A7 27-C4-G7 CC (44 / 100) VH artificial cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TNH11HWVRQAP GQCLEWMGYINPYPGYH AYNE KFQGRATMT SDTSTS TVYME LSSLR SEDTAVYYCARDGYYRDTDVLDYWGQGTL VTVSS 133. BCMA A7 27-C4-G7 CC (44 / 100) VL artifi cial aa DIQMTQSPSSLSASVGDRVTITCQASQDI SNYLNWYQQKPGKAPKLLIYYTSRLHTGV PSRFSGSGSGTDFTFTISSLEPEDIATYY CQQGNTLPWTFGCGTKLEIK 134. BCMA A7 27-C4-G7 CC (44 / 100) scFv artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TNH11HWVRQAP GQCLEWMGYINPYPGYH AYNE KFQGRATMT SDTSTS TVYME LSSLR SEDTAVYYCARDGYYRDTDVLDYWGQGTL VTVSSGGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCQASQDISNYLNWYQQ KPGKAPKLLIYYTSRLHTGVPSRFSGSGS Petition: 870250078518, on September 3, 2025, page. 163 / 222 145 / 188 GTDFTFTISSLEPEDIATYYCQQGNTLPW TFGCGTKLEIK 135. Molécula biespecíf ica BCMA A7 27-C4- G7 CC (44 / 100) x I2C0 artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TNH11HWVRQAP GQCLEWMGYINPYPGYH AYNE KFQGRATMT SDTSTS TVYME LSSLR SEDTAVYYCARDGYYRDTDVLDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCQASQDISNYLNWYQQ KPGKAPKLLIYYTSRLHTGVPSRFSGSGS GTDFTFTISSLEPEDIATYYCQQGNTLPW TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 136.Molécula HLE biespecíf ica BCMA A7 27-C4- G7 CC (44 / 100) x I2C0- scFc artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TNH11HWVRQAP GQCLEWMGYINPYPGYH AYNE KFQGRATMT SDTSTS TVYME LSSLR SEDTAVYYCARDGYYRDTDVLDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCQASQDISNYLNWYQQ KPGKAPKLLIYYTSRLHTGVPSRFSGSGS GTDFTFTISSLEPEDIATYYCQQGNTLPW TFGCGTKVEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP. Petition: 870250078518, on September 3, 2025, page. 164 / 222 146 / 188 GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 137. PM 76- B10.17 CC VH CDR1 artifi cial aa DYYMY 138. PM 76- B10.17 CC VH CDR2 artifi cial aa IISDAGYYTYYSDIIKG Petition: 870250078518, on September 3, 2025, page. 165 / 222 147 / 188 139 PM 76- B10.17 PM 76- B10.17 CC VL CDR3 artifi cial aa QQYDQQLIT 143. PM 76- B10.17 CC VH artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSS 144. PM 76- B10.17 aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIK Petition: 870250078518, on September 3, 2025, page. 166 / 222 148 / 188 146. Molécula compatible ica PM 76-B10.17 CC VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 147 . PM 76- B10.17 CC VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNNYATYYADSVKDRF TISRDSKNTAYLQMNNLKTEDTAVYYCV. Petition: 870250078518, on September 3, 2025, page. 167 / 222 149 / 188 RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGSSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC PM 76- B10.17 CC x I2C0- scFc_delG K artifi cial molecule aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI Petition 870250078518, dated 03 / 09 / 2025, pp. 168 / 222 150 / 188 HLE biespecíf ica TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 149. Molecula bispecíf ica PM 76-B10.17 artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL Petition 870250078518, dated 03 / 09 / 2025, pp. 169 / 222 151 / 188 CC x I2C0 CC (103 / 43)scFc VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 150. PM 76- B10.17 CC x I2C0 CC (103 / 43)scFc molécula HLE biespecíf ica artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL Petition 870250078518, dated 03 / 09 / 2025, pp. 170 / 222 152 / 188 TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEQYGS TYRCVSVLTVLHQDWLNGKEYKCCVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGSGGGGSGGGSGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 151. PM 76- B10.17 CC x I2C0 CC (103 / 43)scFc_delG K HLE bispecific artificial molecule aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDAGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGGSGGGGGSGGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDANVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV Petition 870250078518, dated 03 / 09 / 2025, pages 171 / 222 153 / 188 RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 152. PM 76- B10.11 CC VH CDR1 artificial aa DYYMY 153. PM 76- B10.11 CC VH CDR2 artificial aa IISDGGYYTYYSDIIKG Petition 870250078518, dated 03 / 09 / 2025, pages 172 / 222 154 / 188 154 PM 76- B10.11 PM 76- B10.11 CC VL CDR3 artifi cial aa QQYDQQLIT 158. PM 76- B10.11 CC VH artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSS 159. PM 76- B10.11 CC VL artifi cial aa DIQMTQSPSSLSASVGDRVTITCKASQNV D TNVAWYQQKP GQAPKSLIYSAS YVYWDV PSRFSGSASGTDFTLTISSVQSEDFATYY CQQYDQQLITFGGGTKLEIK 160. PM 76- B10.11 CC scFv artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIK Petition: 870250078518, on September 3, 2025, page. 173 / 222 155 / 188 161. Molécula compatible ica PM 76-B10.11 CC VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 162. PM 76- B10.11 CC VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV. Petition: 870250078518, on September 3, 2025, page. 174 / 222 156 / 188 RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGSSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC PM 76- B10.11 CC x I2C0- scFc_delG K artifi cial molecule aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI Petition 870250078518, dated 03 / 09 / 2025, pp. 175 / 222 157 / 188 HLE biespecíf ica TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 164. Molecula bispecíf ica PM 76-B10.11 artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL Petition 870250078518, dated 03 / 09 / 2025, pp. 176 / 222 158 / 188 CC x I2C0 CC (103 / 43)scFc VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 165. PM 76- B10.11 CC x I2C0 CC (103 / 43) molécula HLE biespecíf ica artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL Petition 870250078518, dated 03 / 09 / 2025, pp. 177 / 222 159 / 188 TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 166. PM 76- B10.11 CC x I2C0 CC (103 / 43) molécula HLE biespecíf ica artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKGLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGGGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV Petition 870250078518, dated 03 / 09 / 2025, pages 178 / 222 160 / 188 RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 167. PM 76- B10.11 CC x I2C0- scFc VH CDR1 artificial aa DYYMY 168. PM 76- B10.11 CC x I2C0- artificial aa IISDGGYYTYYSDIIKG Petition 870250078518, dated 03 / 09 / 2025, pages 179 / 222 161 / 188 scFc VH CDR2 169. PM 76- B10.11 CC x I2C0- scFc VH CDR3 artifi cial aa GFPLLRHGAMDY 170. PM 76- B10.11 CC x I2C0- scFc VL CDR1 artifi cial aa KASQNVD 176-71. B10.11 CC x I2C0- scFc VL CDR2 artifi cial aa SASYVYW 172 . PM 76- B10.11 CC x I2C0- scFc VL CDR3 artifi cial aa QQYDQQLIT 173. PM 76- B10.11 CC x I2C0- scFc VH artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGF SDYYMYWVRQAPGKCLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSS 174 . PM 76- B10.11 CC artifi cial aa DIQMTQSPSSLSASVGDRVTITCKASQNV D TNVAWYQQKP GQAPKSLIYSAS YVYWDV Petition 870250078518, of 03 / 09 / 2025, p. 180 / 222 162 / 188 x I2C0- scFc VL PSRFSGSASGTDFTLTISSVQSEDFATYY CQQYDQQLITFGCGTKLEIK 175. PM 76- B10.11 CC YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIK 176. Molécula bispecíf ica PM 76-B10.11 CC x I2C0-scFc artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL Petition 870250078518, dated 03 / 09 / 2025, pp. 181 / 222 163 / 188 177. PM 76- B10.11 CC VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP Petition 870250078518, dated 03 / 09 / 2025, pp. 182 / 222 164 / 188 HLE spec ica artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG Petition 870250078518, dated 03 / 09 / 2025, pp. 183 / 222 165 / 188 GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 179. Molécula biespecíf ica PM 76-B10.11 CC x I2C0 CC (103 / 43)scFc artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL 180. PM 76- B10.11 CC Petition: 870250078518, on September 3, 2025, page. 184 / 222 166 / 188 (103 / 43) molekula HLE biespecíf ica VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGG KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGGGGSGGGGGSGGGSGGGSGGGSGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPCEQYGSTYRC VSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLPGK Petition 870250078518, de 03 / 09 / 2025, pág. 185 / 222 167 / 188 181. PM 76- B10.11 CC x I2C0 CC (103 / 43) molécula HLE biespecíf ica artifi cial aa QVQLVESGGGLVKPGESLRLSCAASGFTF SDYYMYWVRQAPGKCLEWVAIISDGGYYT YYSDIIKGRFTISRDNAKNSLYLQMNSLK AEDTAVYYCARGFPLLRHGAMDYWGQGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCKASQNVDTNVAWYQQ KPGQAPKSLIYSASYVYWDVPSRFSGSAS GTDFTLTISSVQSEDFATYYCQQYDQQLI TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYCGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQCP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE Petition 870250078518, dated 03 / 09 / 2025, pp. 186 / 222 168 / 188 KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 182. Artificial IgG1 hinge aa DKTHTCPPCP 183. Artificial IgG2 subtype hinge aa ERKCCVECPPCP 184. Artificial IgG3 subtype hinge aa ELKTPLDTTHTCPRCP 185. Artificial IgG3 subtype hinge aa ELKTPLGDTTHTCPRCP 186. Artificial IgG4 subtype hinge aa ESKYGPPCPSCP 187. Artificial G4S ligand aa GGGGS 188. Artificial (G4S)2 ligand aa GGGGSGGGGS 189. Ligand (G4S)3 artificial aa GGGGSGGGGSGGGGS 190. Ligand (G4S)4 artificial aa GGGGSGGGGSGGGGSGGGGS Petition 870250078518, dated 03 / 09 / 2025, pp. 187 / 222 169 / 188 191. Ligand (G4S)5 artifi cial aa GGGGSGGGSGGGGSGGGGSGGGGS 192. Ligand (G4S)6 artifi cial aa GGGGSGGGSGGGGSGGGGGGGSGGGGG S 193. Ligand (G4S) aa7 artifi cial GGGGSGGGSGGGSGGGGSGGGGSGGGG SGGGGS 194. Binder (G4S)8 artifi cial aa GGGGSGGGGSGGGGSGGGGSGGGSGGGGG SGGGGSGGGGS 195. Artificial Peptide Binder 196 GGS GGS Binder. artificial peptide aa PGGDGS 197. Artificial peptide linker aa SGGGGS 198. Artificial peptide linker aa GGGG 199. artificial hexahistidine tag aa HHHHHH 200. Artificial CD3e ligand aa VL QTVVTQEPSLTVSPGGTVTLTCGSSTGAV TSGNYPNWVQQKPGQAPRGLIGGTKFLAP GTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWVFGGGTKLTVL 201. Ligand CD3e VH arti fi cial aa EVQLASVGLSCAGGLASQGLR NSYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKGRFTISRDDSKNTAYLQMNS Petition 870250078518, of 03 / 09 / 2025, p. 188 / 222 170 / 188 LKTEDTAVYYCVRHGNFGNSYVSWWAYWG QGTLVTVSS 202. Ligante CD3e scFv artifi cial aa EVQLVESGGGLVQPGGSLRLSCAASGFTF NSYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKGRFTISRDDSKNTAYLQMNS MU 8-B7 CC I2C0-scFc VH CDR1 MU 8-B7 CC x I2C0-scFc VH CDR2 8-B7 CC x I2C0-scFc VH CDR2 8-B7 CC x DIDASGSTKYNPSLKS 206. MU 8-B7 CC I2C0-scFc VL CDR1 artificial aa SGDKLGDKYAS 207. MU 8-B7 CC x artificial aa QDRKRPS Petition: 870250078518, on September 3, 2025, page. 189 / 222 171 / 188 l2C0-scFc VL CDR2 208. MU 8-B7 CC x I2C0-SCFC VL CDR3 artificial aa QAWGSSTAV 209. MU 8-B7 CC x I2C0-SCFC VH artificial aa QVQLQQWGAGLLKPSETLSLTCAVYGGSF SGYYWSWIRQPPGKCLEWIGDIDASGSTK YNP S LKSRVTISLDTSKNQF S LKLNSVTA ADTAVYFCARKKYSTVWSYFDNWGQGTLV TVSS 210. MU 8-B7 CC x I2C0-SCFC VL artificial aa SYELTQPSSVSVPPGQTASITCSGDKLGD KYASWYQQKP GQSPVLVIYQDRKRPSGVP ERFSGSNSGNTATLTISGTQAMDEADYYC QAWGSSTAVFGCGTKLTVL 211. MU 8-B7 CC x I2C0-SCFC ScFv artifi cial aa QVQLQQWGAGLLKPSETLSLTCAVYGGSF SGYYWSWIRQPPGKCLEWIGDIDASGSTK YNP S LKSRVTISLDTSKNQF S LKLNSVTA ADTAVYFCARKKYSTVWSYFDNWGQGTLV TVSSGGGGSGGGGSGGGGSSYELTQPSSV SVPPGQTASITCSGDKLGDKYASWYQQKP GQSPVLVIYQDRKRPSGVPERFSGSNSGN TATLTISGTQAMDEADYYCQAWGSSTAVF GCGTKLTVL 212 . Molécula biespecíf ica MU 8- B7 CC x I2C0-scFc artifi cial aa QVQLQQWGAGLLKPSETLSLTCAVYGGSF SGYYWSWIRQPPGKCLEWIGDIDASGSTK YNP S LKSRVTISLDTSKNQF S LKLNSVTA ADTAVYFCARKKYSTVWSYFDNWGQGTLV TVSSGGGGSGGGGSGGGGSSYELTQPSSV SVPPGQTASITCSGDKLGDKYASWYQQKP Petition 870250078518, dated 03 / 09 / 2025, pp. 190 / 222 172 / 188 GQSPVLVIYQDRKRPSGVPERFSGSNSGN TATLTISGTQAMDEADYYCQAWGSSTAVF GCGTKLTVLSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRH GNFGNSYISYWAYWGQGTLVTVSSGGGGS GGGGSGGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVL 213. Molécula HLE biespecíf ica MU 8- B7 CC x I2C0-scFc Artifi cial aa QVQLQQWGAGLLKPSETLSLTCAVYGGSF SGYYWSWIRQPPGKCLEWIGDIDASGSTK YNP S LKS RVTISLDTSKNQF S LKLNSVTA ADTAVYFCARKKYSTVWSYFDNWGQGTLV TVSSGGGGSGGGGSGGGGSSYELTQPSSV SVPPGQTASITCSGDKLGDKYASWYQQKP GQSPVLVIYQDRKRPSGVPERFSGSNSGN TATLTISGTQAMDEADYYCQAWGSSTAVF GCGTKLTVLSGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRH GNFGNSYISYWAYWGQGTLVTVSSGGGGS GGGGSGGGGSQTVVTQEPSLTVSPGGTVT LTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVLGGGGDKTHTCPPCPAPELLGGPSVFL Petition 870250078518, of 03 / 09 / 2025, p. 191 / 222 173 / 188 FPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPCEEQYGSTY RCVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKKQPREPQVYTLPPPSREEM TKNQVSLTCLVKGFYPSDIAVEWNGQ ENNYCTTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGSGGGGGGGGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDVFKFK NWYVDGVEVHNACTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVKSRWQQQQQ NVFSCSVMHEALHNHYTQKSLSLSPGK 214 . CL-1 VH CDR1 Artifi cial aa GYYMH 215. CL-1 VH CDR2 Artifi cial aa WINPNSGGTKYAQKFQG 216. CL-1 VH CDR3 Artifi cial aa DRITVAGTYYYYGMDV 217 . CL-1 VL CDR1 Artifi cial aa RASQGVNNWLA 218. CL-1 VL CDR2 Artifi cial aa TASSLQS 219. CL-1 VL CDR3 Artifi cial aa QQANSFPIT TGYYMHWVRQAPGQCLEWMGWINPNSGGT Petition 870250078518, of 03 / 09 / 2025, p. 192 / 222 174 / 188 KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSS 221. CL-1 VL Artifi cial aa DIQMTQSPSSVSASVGDRVTITCRASQGV NNWLAWYQQKP GKAP KLLIYTASSLQSGV PSRFSGSGSGTDFTLTIRSLQPEDFATYY CQQANSFPITFGCGTRLEIK 222. CL-1 scFv Artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIK 223. CL-1 x I2C molécula 224. biespecíf ica Artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSV Petition: 870250078518, on September 3, 2025, page. 193 / 222 175 / 188 KDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL 225. Molécula biespecíf ica CL-1 x I2C- 6His etiqueta -his Artifi cial aa QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGGSGGGGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFLTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSV KDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLHHHHHH 226. TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGGSGGGGSGGGGSDIQMT Petition 870250078518, de 03 / 09 / 2025, pág. 194 / 222 176 / 188 x I2C- scFc QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYADSV KDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLGGGGDKTHTCPPPAPELLG GPSVFLFPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPECEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPPVLDSDGSFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPKGGGGSGGGGSGGGGSGGGSG GGGSGGGGSDKTHTCPPPCAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEKYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Petition: 870250078518, on September 3, 2025, page. 195 / 222 177 / 188 227. CL-2 VH CDR1 Artificial aa GYYMH 228. CL-2 VH CDR2 Artificial aa WINPNSGGTKYAQKFQG 229. CL-2 VH CDR3 Artificial aa DRITVAGTYYYYGMDV 230. CL-2 VL CDR1 Artificial aa RASQGVNNWLA 231. CL-2VL CDR2 Artificial aa TASSLQS 232. CL-2 VL CDR3 Artificial aa QQANSFPIT 233. CL-2 VH Artificial aa QVQMVQSGAEVKKHGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSS 234. CL-2 VL Artificial aa DIQMTQSPSSVSASVGDRVTITCRASQGV NNWLAWYQQKP GKAP KLLIYTASSLQSGV PSRFSGSGSGTDFTLTIRSLQPEDFATYY CQQANSFPITFGCGTRLEIK 235. CL-2 scFv Artificial aa QVQMVQSGAEVKKHGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIK Petition: 870250078518, on September 3, 2025, page. 196 / 222 178 / 188 236. molécula biespecíf ica CL-2 x I2C Artifi cial aa QVQMVQSGAEVKKHGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSV KDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL 237.Molécula biespecíf ica CL-2 x I2C- 6His etiqueta -his Artifi cial aa QVQMVQSGAEVKKHGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFTLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNNYATYYADSV KDRFTISRDSKNTAYLQMNNLKTEDTAV. Petition: 870250078518, on September 3, 2025, page. 197 / 222 179 / 188 YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLHHHHHH 238. Molécula scFc biespecíf ica CL-2 x I2C- scFc Artifi cial aa QVQMVQSGAEVKKHGASVKVSCKASGYTF TGYYMHWVRQAPGQCLEWMGWINPNSGGT KYAQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDRITVAGTYYYYGMDVWG QGTTVTVSSGGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQGVNNWLA WYQQKPGKAPKLLIYTASSLQSGVPSRFS GSGSGTDFLTIRSLQPEDFATYYCQQAN SFPITFGCGTRLEIKSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSV KDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSTVVTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLGGGGDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKL Petition: 870250078518, on September 3, 2025, page. 198 / 222 180 / 188 TVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKA CD70 HCDR1 Artifi 240. social aa AISGSGGRTFYAESVEG 241. CD70 HCDR3 Artificial aa HDYSNYPYFDY 242 . CD70 LCDR1 Artificial aa RASQSVRSTYLA 243. CD70 LCDR2 Artificial aa GASSRAT 244. CD70 LCDR3 Artificial hair removal device QQYGDLPFT 245. CD70 VH Artificial hair removal device EVQLLESGGGMVQPGGSLRLSCAASGFTF STYAMSWVRQAPGKCLEWVSAISGSGGRT FYAESVEGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKHDYSNYPYFDYWGQGTLV TVSS 246. CD70 VL Artificial hair removal device EIVLTQSPGTLSLSPGERATLSCRASQSV RSTYLAWYQQKPGQAPRLLIYGASSRATG Petition: 870250078518, on September 3, 2025, page. 199 / 222 181 / 188 IPDRFSGSGSGTDFTLTISRLEPEDFAVY SCQQYGDLPFTFGCGTKLEIK 247 . CD70 VHVL Artifi cial aa EVQLLESGGGMVQPGGSLRLSCAASGFTF STYAMSWVRQAPGKCLEWVSAISGSGGRT FYAESVEGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKHDYSNYPYFDYWGQGTLV TVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRASQSVRSTYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYSCQQYGDLPF TFGCGTKLEI 248. CD70 x I2C Artifi cial aa EVQLLESGGGMVQPGGSLRLSCAASGFTF STYAMSWVRQAPGKCLEWVSAISGSGGRT FYAESVEGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKHDYSNYPYFDYWGQGTLV TVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRASQSVRSTYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYSCQQYGDLPF TFGCGTKLEIKSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSSGGG GSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSST GAVT S GNYPNWVQQKP GQAP RGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTV Petição 870250078518, de 03 / 09 / 2025, pág. 200 / 222 182 / 188 249. Fc monomer 1 + c / -g Artificial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 250. Fc monomer 2 + c / g / delGK Artificial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP 251. Fc-c / +g Artificial monomer-3 aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 252.monomer- 4 of Fc c / +g / delG K Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEWKFNWY VDGVEVHNACTKPREEQYNSTYRVVSVLT VLHQDWLNGKE YPACKVSNKAL SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT. Petition 870250078518, of 03 / 09 / 2025, p. 201 / 222 183 / 188 PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP 253. Fc-c / -g Artificial monomer aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYGSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 254 . Fc-6 monomer -c / g / delGK Artificial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYGSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP 255. Fc+7 monomer +c / +g Artificial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYNSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 256.monomer- 8 of Fc +c / +g / del GK Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEWKFNWY VDGVEVHNACTKPCEEQYNSTYRCVSVLT VLHQDWLNGKE YPACKVSNKALTINK. Petition 870250078518, of 03 / 09 / 2025, p. 202 / 222 184 / 188 SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP 257. scFc-1 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYGSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 258. scFc-2 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSDKTHTC Petição 870250078518, de 03 / 09 / 2025, pág. 203 / 222 185 / 188 PPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPCEEQYGSTYRCVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQ PREP QVYTLP P S REEMTKNQVSLTC LVKG FYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSP 259. scFc-3 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 260. scFc-4 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL Petição 870250078518, de 03 / 09 / 2025, pág. 204 / 222 186 / 188 TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQ PREP QVYTLP P S RE EMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSP 261. scFc-5 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYGSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYGSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Petition 870250078518, dated 03 / 09 / 2025, pp. 205 / 222 187 / 188 262. scFc-6 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYGSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYGSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQ PREP QVYTLP P S RE EMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSP 263. scFc-7 Artifi cial aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYNSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYNSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAK Petição 870250078518, de 03 / 09 / 2025, pág. 206 / 222 188 / 188 GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 264. scFc-8 Artifi cial Aa DKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYNSTYRCVSVLT VLHQDWLNGKE YKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPCEEQYNSTYRCVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQ PREP QVYTLP P S RE EMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSP

Claims

1. Method for preparing a lyophilized bispecific molecule composition, wherein the method is characterized in that it comprises (a) inducing ice nucleation in a liquid bispecific molecule composition having a concentration of a bispecific molecule of at least about 10 mg / ml in a flask exposed to a first temperature of about 18 °C to about -10 °C for a first period of time of about 60 minutes to about 270 minutes; (b) exposing the flask from step (a) to a second temperature of about -25 °C to -50 °C for a second period of time of about 1 hour to about 5 hours; (c) drying the composition from step (b) at a third temperature of about -5 °C to about -25 °C for a third period of time of about 25 hours to about 70 hours;and (d) further drying the composition from step (c) at a fourth temperature of about 25 °C to about 50 °C for a fourth period of time of about 4 hours to about 12 hours, resulting in a vial comprising a lyophilized bispecific molecule composition having a percentage content of high molecular weight species (HMWS) less than or equal to 1.5% (w / v), wherein the bispecific molecule comprises at least three domains, wherein: • the first domain binds to a tumor antigen on a target cell; • the second domain binds to an extracellular epitope of the human and / or Macaca CD3e chain; and Petition 870250078518, dated 03 / 09 / 2025, p. 208 / 222 2 / 9 • the third domain is fused to the second domain by a peptide linker, wherein the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain, wherein said two polypeptide monomers are fused to each other by means of a peptide linker.

2. Method according to claim 1, characterized in that step (a) comprises exposing the flask to the first temperature for about 90 minutes to about three hours.

3. Method according to claim 2, characterized in that step (a) comprises exposing the flask to the first temperature for about 90 minutes to about two hours, preferably 110 minutes.

4. Method, according to any one of claims 1 to 3, characterized in that the first temperature in step (a) is from about -12 °C to about -17 °C.

5. Method according to claim 4, characterized in that the first temperature in step (a) is about -15 °C.

6. Method according to any one of claims 1 to 5, characterized in that step (a) further comprises maintaining the nucleated protein composition on ice at the first temperature for a post-nucleation time period of up to 90 minutes.

7. Method according to claim 6, characterized in that the post-nucleation time period is from about 20 minutes to about 90 minutes. Petition 870250078518, dated 03 / 09 / 2025, pp. 209 / 222 3 / 9 8. Method according to claim 7, characterized in that the post-nucleation time period is about 30 minutes.

9. Method according to any one of claims 1 to 8, characterized in that step (b) comprises exposing the flask to a second temperature of about -45 °C.

10. Method, according to any one of claims 1 to 9, characterized in that the second time period is from about two hours to about four hours.

11. Method according to claim 10, characterized in that the second time period is about three hours.

12. Method, according to any one of claims 1 to 11, characterized in that the first temperature is transitioned to the second temperature at a rate of about 0.01 °C per minute to about 0.5 °C per minute.

13. Method according to claim 12, characterized in that the first temperature is transitioned to the second temperature at a rate of about 0.2 °C per minute.

14. Method, according to any one of claims 1 to 13, characterized in that the third temperature in step (c) is from about 0 °C to about -20 °C.

15. Method according to claim 14, characterized in that the third temperature of step (c) is from about -5 °C to about -10 °C.

16. Method according to claim 15, characterized in that the third temperature in step (c) is about -8 °C. Petition 870250078518, dated 03 / 09 / 2025, pp. 210 / 222 4 / 9 17. Method according to any one of claims 1 to 9, characterized in that step (c) comprises exposing the flask comprising the bispecific molecule composition to an increase in temperature at a rate of about 0.01 °C per minute to about 0.5 °C per minute.

18. Method according to any one of claims 1 to 17, characterized in that the transition from step (b) to step (c) comprises increasing the temperature at a rate of about 0.2 °C per minute to about 0.7 °C per minute and maintaining the flask at a temperature of about -40 °C to about -30 °C for about 15 minutes to about one hour.

19. Method according to any one of claims 1 to 18, characterized in that the liquid bispecific molecule composition is exposed in step (a) to a first temperature of about -15 °C for about 110 minutes plus about 30 minutes after nucleation, is exposed in step (b) to a second temperature of about -45 °C for about 3 hours; the composition from step (b) is dried in step (c) at a third temperature of about -8 °C for about 50 hours; and the composition from (c) is further dried at a fourth temperature of about 40 °C for about 8 hours.

20. Method, according to any one of claims 1 to 18, characterized in that the bispecific molecule is a single-chain molecule.

21. Method, according to any one of claims 1 to 18, characterized in that the bispecific molecule is present in the composition at a concentration of about 10 mg / ml to about 30 mg / ml or about 20 mg / ml to about 30 mg / ml, preferably about 15 mg / ml to about 25 mg / ml.

22. Method, according to any one of claims 1 to 18, characterized in that the method results in a vial comprising a lyophilized bispecific molecule composition that shows aggregation in terms of formation of high molecular weight species (HMSW) less than or equal to about 1.5% (w / v) or preferably less than or equal to about 11% (w / v).

23. Method, according to any one of claims 1 to 22, characterized in that said third domain comprises an amino-to-carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

24. Method, according to any one of claims 1 to 23, characterized in that each of said polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 249-256 or identical to a sequence selected from the group consisting of: SEQ ID NO: 249-256.

25. Method, according to any one of claims 1 to 24, characterized in that the first domain binds to CD33, CDH19, MSLN, FLT3, BCMA, CD19, MUC17, CDH3, CLDN18.2, CD70, EGFRviii, EpCAM, DLL3 and / or PSMA.

26. Method, according to any one of claims 1 to 25, characterized in that the first binding domain of the bispecific molecule 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: Petition 870250078518, dated 03 / 09 / 2025, p. 212 / 222 6 / 9 (a) CDR-H1 as represented in SEQ ID NO: 4, CDRH2 as represented in SEQ ID NO: 5, CDR-H3 as represented in SEQ ID NO: 6, CDR-L1 as represented in SEQ ID NO: 1, CDR-L2 as represented in SEQ ID NO: 2 and CDR-L3 as represented in SEQ ID NO: 3, (b) CDR-H1 as represented in SEQ ID NO: 29, CDR-H2 as represented in SEQ ID NO: 30, CDR-H3 as represented in SEQ ID NO: 31, CDR-L1 as represented in SEQ ID NO: 34, CDR-L2 as represented in SEQ ID NO: 35 and CDR-L3 as represented in SEQ ID NO: 36, (c) CDR-H1 as represented in SEQ ID NO: 42,CDR-H2 as represented in SEQ ID NO: 43, CDR-H3 as represented in SEQ ID NO: 44, CDR-L1 as represented in SEQ ID NO: 45, CDR-L2 as represented in SEQ ID NO: 46 and CDR-L3 as represented in SEQ ID NO: 47, (d) CDR-H1 as represented in SEQ ID NO: 53, CDR-H2 as represented in SEQ ID NO: 54, CDR-H3 as represented in SEQ ID NO: 55, CDR-L1 as represented in SEQ ID NO: 56, CDR-L2 as represented in SEQ ID NO: 57 and CDR-L3 as represented in SEQ ID NO: 58, (e) CDR-H1 as represented in SEQ ID NO: 65, CDR-H2 as represented in SEQ ID NO: 66, CDR-H3 as represented in SEQ ID NO: 67, CDR-L1 as represented in SEQ ID NO: 68, CDR-L2 as represented in SEQ ID NO: 69 and CDR-L3 as represented in SEQ ID NO: 70, Petition 870250078518, dated 03 / 09 / 2025, page 213 / 222 7 / 9 (f) CDR-H1 as represented in SEQ ID NO: 83, CDR-H2 as represented in SEQ ID NO: 84,CDR-H3 as depicted in SEQ ID NO: 85, CDR-L1 as depicted in SEQ ID NO: 86, CDR-L2 as depicted in SEQ ID NO: 87 and CDR-L3 as depicted in SEQ ID NO: 88, (g) CDR-H1 as depicted in SEQ ID NO: 94, CDR-H1 as depicted in SEQ ID NO: 94, CDR-H1 as depicted in SEQ ID NO: 94, CDR-H1 as depicted in SEQ ID NO: 95: CDR-H3 as depicted in SEQ ID NO: 96, CDR-L1 as depicted in SEQ ID NO: 97, CDR-L2 as depicted in SEQ ID NO: 98 and CDR-L3 as depicted in SEQ ID NO: 99, (h) CDR-H1 as depicted in SEQ ID NO: 105, CDR-H1 as depicted in SEQ ID NO: 105, CDR-H1 as depicted in SEQ ID NO: 105, CDR-H1 as depicted in SEQ ID NO: 92 106, CDR-H3 as depicted in SEQ ID NO: 107, CDR-L1 as depicted in SEQ ID NO: 109, CDR-L2 as depicted in SEQ ID NO: 110 and CDR-L3 as depicted in SEQ ID NO: 111, (i) CDR-H1 as depicted in SEQ ID NO: 12, (i) CDR-H1 as depicted in SEQ ID NO: 12 SEQ ID NO: 116, CDR-H3 as depicted in SEQ ID NO: 117, CDR-L1 as depicted in SEQ ID NO: 118,CDR-L2 as represented in SEQ ID NO: 119 and CDR-L3 as represented in SEQ ID NO: 120, (j) CDR-H1 as represented in SEQ ID NO: 126, CDR-H2 as represented in SEQ ID NO: 127, CDR-H3 as represented in SEQ ID NO: 128, CDR-L1 as represented in SEQ ID NO: 129, CDR-L2 as represented in SEQ ID NO: 130 and CDR-L3 as represented in SEQ ID NO: 131, Petition 870250078518, dated 03 / 09 / 2025, page. 214 / 222 8 / 9 (k) CDR-H1 as represented in SEQ ID NO: 137, CDR-H2 as represented in SEQ ID NO: 138, CDR-H3 as represented in SEQ ID NO: 139, CDR-L1 as represented in SEQ ID NO: 140, CDR-L2 as represented in SEQ ID NO: 141 and CDR-L3 as represented in SEQ ID NO: 142, (l) CDR-H1 as represented in SEQ ID NO: 152, CDR-H2 as represented in SEQ ID NO: 153, CDR-H3 as represented in SEQ ID NO: 154, CDR-L1 as represented in SEQ ID NO: 155,CDR-L2 as represented in SEQ ID NO: 156 and CDR-L3 as represented in SEQ ID NO: 157, (m) CDR-H1 as represented in SEQ ID NO: 167, CDR-H2 as represented in SEQ ID NO: 168, CDR-H3 as represented in SEQ ID NO: 169, CDR-L1 as represented in SEQ ID NO: 170, CDR-L2 as represented in SEQ ID NO: 156 represented in SEQ ID NO: 171 and CDR-L3 as represented in SEQ ID NO: 172, (n) CDR-H1 as represented in SEQ ID NO: 203, CDR-H2 as represented in SEQ ID NO: 204, CDR-H3 as represented in SEQ ID NO: 205, CDR-L1 as represented in SEQ ID NO: 206, CDR-L2 as represented in SEQ ID NO: 207 and CDR-L3 as per represented in SEQ ID NO: 208; (o) CDR-H1 as represented in SEQ ID NO: 214, CDR-H2 as represented in SEQ ID NO: 215, CDR-H3 as represented in SEQ ID NO: 216, CDR-L1 as represented in SEQ ID NO: 217, CDR-L2 as represented in SEQ ID NO: 218 and CDR-L3 as represented in SEQ ID NO: 219; Petition 870250078518,of 03 / 09 / 2025, p. 215 / 222 9 / 9 (p) CDR-H1 as depicted in SEQ ID NO: 226, CDR-H2 as depicted in SEQ ID NO: 227, CDR-H3 as depicted in SEQ ID NO: 229, CDR-L1 as depicted in SEQ ID NO: 229, CDR-L1 as depicted in SEQ ID NO: 229, CDR-L as depicted in SEQ ID NO: 230 CDR-L3 conforming depicted in SEQ ID NO: 231; and (q) CDR-H1 as depicted in SEQ ID NO: 238, CDR-H2 as depicted in SEQ ID NO: 239, CDR-H3 as depicted in SEQ ID NO: 240, CDR-L1 as depicted in SEQ ID NO: 241, CDR-L2 as depicted in SEQ ID NO: 23 and CDR-L2 as depicted in SEQ ID NO: 23 NO: 243., 27. Method, according to any one of claims 1 to 26, characterized in that the first domain has an amino acid sequence selected from the group consisting of the following 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, 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, 181, 223, 235 and 246, preferably 100 to 104.

28. A method, according to any one of claims 1 to 27, characterized in that ice nucleation is induced by means of ice fog or depressurization.

29. Lyophilized bispecific molecule composition characterized in that it is prepared by means of the method as defined in any one of claims 1 to 28.

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