Humanized rodent her2 sequence

Abstract

The presently-disclosed subject matter relates to humanized rodent HER2 sequences. Embodiments of the presently-disclosed subject matter include polypeptides and nucleic acids, cells comprising such polypeptides and such nucleic acids, and genetically-modified rodents. Embodiments of the presently-disclosed subject matter include methods for assessing a candidate therapeutic that contains trastuzumab, pertuzumab, derivatives thereof, and / or drug conjugates thereof.

Description

HUMANIZED RODENT HER2 SEQUENCE byJustin M. Balko Julia SteeleAssignee: Vanderbilt UniversityAttorney Docket No.: 11672N / 24103WORELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 621,888 filed January 17, 2024, the entire disclosure of which is incorporated herein by this reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (Balko VU24103 Sequence Listing.xml; Size: 59,073 bytes; and Date of Creation: January 15, 2025) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] The presently-disclosed subject matter generally relates to a humanized rodent HER2 sequence. Certain embodiments of the presently-disclosed subject matter relate to polypeptides, nucleic acids, cells comprising such nucleic acids and expressing such polypeptides, and genetically-modified rodents. Certain embodiments of the presently- disclosed subject matter relate to using such sequences, cells, and genetically-modified mice and rats to assess a candidate therapeutic that contains trastuzumab, pertuzumab, or derivatives thereof.INTRODUCTION

[0004] Trastuzumab and pertuzumab are monoclonal antibodies that have been useful in the treatment of HER2 -positive cancers, a subset of aggressive cancers characterized byoverexpression of the HER2 receptor. HER2, also known as ERBB2, refers to the human epidermal growth factor receptor 2 protein.

[0005] Trastuzumab works by binding to the extracellular domain (subdomain IV) of HER2, blocking receptor activation and downstream signaling pathways that drive tumor growth and survival. In this manner, trastuzumab inhibits the proliferation of cells that overexpress HER2. It also recruits immune cells to attack HER2-expressing tumor cells through antibody-dependent cellular cytotoxicity (ADCC) and inhibits HER2 shedding, which could otherwise promote immune evasion, thereby recruiting the immune system to attack the cancer cells.

[0006] Pertuzumab, on the other hand, binds to a different part of the HER2 receptor (subdomain II), preventing its dimerization with other HER family receptors, such as HER3. This dual blockade enhances the inhibition of HER2-driven signaling when trastuzumab and pertuzumab are combined, providing a more comprehensive therapeutic approach.

[0007] Trastuzumab, also know n by the brand name Herceptin®, is used for treatment in connection with a number of HER2-positive cancers, including breast and advanced gastric cancers. In early-stage breast cancer, it is used in combination with chemotherapy for neoadjuvant and adjuvant therapy to shrink tumors or reduce recurrence risk. It also plays a role in metastatic breast cancer, either as monotherapy or in combination with other agents.

[0008] Pertuzumab, also known by the brand name Peijeta®, has been used in combination with trastuzumab and chemotherapy to enhance efficacy. This regimen is used for HER2-positive metastatic breast cancer and is also employed in neoadjuvant and adjuvant settings. Together, trastuzumab and pertuzumab improve progression-free and overall survival in patients with HER2 -positive cancers.

[0009] Trastuzumab and pertuzumab have been used to treat various HER2 -positive cancers, with their primary' indications being breast cancer and advanced gastric or gastroesophageal junction adenocarcinoma. In breast cancer, both agents are used across early-stage and metastatic settings. In early-stage HER2 -positive breast cancer, trastuzumab and pertuzumab are combined with chemotherapy as neoadjuvant (pre-surgery) or adjuvant (post-surgery ) therapies to shrink tumors and reduce recurrence risk. In metastatic HER2- positive breast cancer, the combination of trastuzumab, pertuzumab, and chemotherapy has demonstrated improvements in overall and progression-free survival. Trastuzumab is alsoapproved for HER2-positive advanced gastric or gastroesophageal junction adenocarcinoma when combined with chemotherapy. Beyond these indications, trastuzumab has been investigated in other HER2 -positive cancers, including metastatic colorectal cancer, HER2- positive non-small cell lung cancer (NSCLC), uterine serous carcinoma, and rare cancers like salivary gland tumors. Pertuzumab’s role outside of breast cancer continues to be explored.

[0010] More recent developments related to HER2-targeted therapies have included the creation of antibody -drug conjugates (ADCs). These complex molecules combine antibodies like trastuzumab with a therapeutic agent, such as a chemotherapeutic or immunotherapeutic drug. The targeting ability of trastuzumab allows these agents to be delivered directly to HER2 -positive cancer cells, increasing efficacy while minimizing systemic side effects. Examples include trastuzumab emtansine (T-DM1), a conjugate of trastuzumab and the chemotherapeutic agent DM1, and trastuzumab deruxtecan, which links trastuzumab to a cytotoxic agent called deruxtecan. These ADCs are particularly effective in treating HER2- positive metastatic breast cancer, where their targeted approach offers significant advantages over traditional chemotherapy. Meanwhile, pertuzumab, another HER2-targeting monoclonal antibody, is often used in combination with trastuzumab and chemotherapy to enhance the therapeutic blockade of HER2. Although pertuzumab is not currently linked to an antibodydrug conjugate, its synergistic role alongside trastuzumab provides a foundation for exploring new combination strategies with ADCs.

[0011] As the development of new trastuzumab-based and HER2-targeted drug conjugates advances, preliminary efficacy testing remains an important step before initiating clinical trials with human subjects. Early evaluations typically involve assessing the conjugate’s ability to specifically target and eliminate HER2 -positive cancer cells in vitro using cultured cell lines. Pertuzumab, when used in preclinical models alongside trastuzumab-based conjugates, may also provide insights into how dual HER2 blockade influences conjugate performance. However, transitioning from cell culture studies to animal models presents challenges, including ensuring that the conjugate maintains its specificity and efficacy in more complex biological systems. Further exploration of combined HER2- targeted strategies could pave the way for more effective treatments in both preclinical and clinical settings.

[0012] Currently, there is a significant limitation in the availability of orthotopic, immunocompetent rodent tumor models that express functional HER2 protein capable ofbinding trastuzumab. This constraint necessitates reliance on either primate models or immune-deficient mouse models, each with inherent drawbacks. Primate models, while immunocompetent, are costly and logistically challenging, whereas immune-deficient mouse models lack clinical translatability due to their inability to replicate a functional immune system.

[0013] Some biotechnology companies have developed licensable humanized HER2 mouse models in which the endogenous mouse HER2 locus is replaced with a chimeric gene encoding the human HER2 extracellular domain fused to the mouse intracellular domain. While promising, this approach has limitations. The human extracellular domain is large and complex, making it difficult to modify. Additionally, if such a chimeric sequence were transduced into cells and implanted into a naive mouse, it could elicit an immune rejection response. Nevertheless, these mice could tolerate similar sequences when carried in human cell lines, albeit requiring a multi-pronged experimental approach.

[0014] A synthetic HER2 protein has also been developed1that incorporates the trastuzumab-binding human HER2 extracellular domain fused to the transmembrane domain of mouse HER2. However, this synthetic receptor is non-functional and acts solely as a decoy to direct trastuzumab to specific cell targets. While useful in certain experimental contexts, this design lacks the signaling capabilities necessary for studying HER2-driven oncogenesis or evaluating the full therapeutic potential of trastuzumab-based treatments.

[0015] Accordingly, there remains an unmet need for advanced, efficient, and clinically relevant methodologies to evaluate trastuzumab and pertuzumab-based treatments.SUMMARY

[0016] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0017] This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary' is merely exemplary' of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, thosefeatures can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary’ or not. To avoid excessive repetition, this Summary’ does not list or suggest all possible combinations of such features.

[0018] Humanized rodent receptors or chimeric human-rodent receptors are useful tools in drug development because they mimic human biology by expressing human receptor proteins, making these rodent models more representative of human physiological responses. This allows researchers to evaluate drug interactions, including efficacy, pharmacodynamics, and safety, in a preclinical setting. Such models reduce the need for early testing in humans or non-human primates, offering significant ethical and practical benefits. By facilitating early-stage drug testing, humanized rodent receptors help identify potential drug candidates and assess adverse effects earlier in the development pipeline.

[0019] Beyond drug development, the nucleotide sequence encoding these humanized receptors is useful in gene therapy research for delivering or replacing dysfunctional genes, studying receptor variants associated yvith diseases, and conducting in vitro studies to understand molecular mechanisms. The polypeptide sequence of the humanized receptor aids in producing purified proteins for structural studies, drug screening, and generating therapeutic agents like antibodies. Genetically modified mice or rats expressing humanized receptors allow researchers to create disease models.

[0020] Trastuzumab and pertuzumab do not bind to rodent HER2.5The presently- disclosed subject matter helps meet the need for advanced, efficient, and clinically relevant methodologies to evaluate trastuzumab and pertuzumab-based treatments. Such models alloyv for testing of trastuzumab and pertuzumab-based treatments, including antibody-drug conjugates and antibody derivatives. Such testing can include therapeutic efficacy and mechanisms of action in immunocompetent systems, ultimately improving the translational potential of preclinical findings.

[0021] The presently-disclosed subject matter relates to a chimeric rodent-human Her2 receptor. Relative to the amino acid sequence of the rodent Her2 proteins, strategic substitutions of specific amino acids are provided in the trastuzumab- and / or pertuzumab- binding regions from the human HER2 amino acid sequence. Rodent-human chimeric sequences disclosed herein are capable of binding trastuzumab, pertuzumab, trastuzumab- emtansine, trastuzumab-deruxtecan, and biosimilars.

[0022] The sequences disclosed herein can be transduced into mouse cells, rat cells, cancer cells, or used to modify a mouse or rat genome. Transduced cell lines can be orthotopically injected into immunocompetent mice or rats without immune rejection.

[0023] The resulting cell line or rodent has a primarily rodent Her2 receptor that can be targeted with trastuzumab, pertuzumab, or similarly-binding antibodies, or antibody-drug conjugates in vitro or in vivo. The retention of the rodent portion of the sequence ensures minimal sequence deviation from the rodent, permitting studies in immunocompetent rodents, as a fully human sequence or substantially human sequence would be rejected by the rodent immune system.

[0024] The sequences disclosed herein, when placed for example in a tumor cell line and implanted into an immunocompetent host rodent, permits the use of trastuzumab, pertuzumab, or antibody drug conjugates to study mechanistic function, contribution and adaptation of the immune system to therapy, novel combinations of therapeutics, and identification of mechanisms of resistance and biomarkers thorough studies like CRISPR / Cas9 genetic knockout screens.

[0025] The presently-disclosed subject matter includes humanized rodent HER2 sequences. In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 1 with at least three amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 2 with at least six nucleic acid modifications.

[0026] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 3 In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 4.

[0027] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 5 In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 6.

[0028] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 7 with at least two amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 8 with at least four nucleic acid modifications.

[0029] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 10.

[0030] In an embodiment of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 or a fragment thereof comprising at least amino acid residues 580-628, in which 3-7 amino acids have been modified, wherein the amino acid modifications comprise S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S580P, E586V, Y617F, and 1623 A.

[0031] In another embodiment, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 or a fragment thereof comprising at least amino acid residues 253-343, in which 2 amino acids have been modified, wherein the amino acid modifications comprise P318L and N319H.

[0032] In another embodiment, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 or a fragment thereof comprising at least amino acid residues 253-343 and 580-628, in which 5-9 amino acids have been modified, wherein the amino acid modifications comprise P318L, N319H, S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S580P, E586V, Y617F, and 1623 A.

[0033] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 or a fragment thereof comprising at least nucleotides 1738- 1884, in which 6-13 nucleotides have been modified, wherein the nucleotide modifications comprise t!780c. a!782c, t!783c, t!785c, cl787t, and c!838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tl738c, gl740c, a!757t, a!850t, cl 85 It, al 867g, and t!868c.

[0034] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 or a fragment thereof comprising at least nucleotides 757-1011, in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise.

[0035] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 or a fragment thereof comprising at least nucleotides 757-1011and 1738-1884, in which 10-17 nucleotides have been modified, wherein the nucleotide modifications comprise c953t, g954t, a955c, and c957t, tl780c. al782c, tl783c, tl785c, cl787t, and cl 838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tl738c, gl740c, al757t, al850t, cl 851t, al867g, and tl868c.

[0036] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 13 with at least three amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 14 with at least four nucleic acid modifications.

[0037] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 16.

[0038] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 17 with at least three amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 18 with at least five nucleic acid modifications.

[0039] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 20.

[0040] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 21 with at least two amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 22 with at least three nucleic acid modifications.

[0041] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 24.

[0042] In another embodiment, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 25 or a fragment thereof comprising at least amino acid residues583-631, in which 3 amino acids have been modified, wherein the amino acid modifications comprise S597P. S598P. and S599F.

[0043] In another embodiment, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 25 or a fragment thereof comprising at least amino acid residues 256-346, in which 2 amino acids have been modified, wherein the amino acid modifications comprise P321L and N322H.

[0044] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 or a fragment thereof comprising at least nucleotides 1747- 1894, in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise tl789c, al 791c, tl 792c, cl 796t, and cl797t.

[0045] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 or a fragment thereof comprising at least nucleotides 766-1040, in which 3 nucleotide have been modified, wherein the nucleotide modifications comprise c962t, g963t, and a964c.

[0046] In another embodiment, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 or a fragment thereof comprising at least nucleotides 766-1040 and 1747-1894, in which 8nucleotides have been modified, wherein the nucleotide modifications comprise c962t, g963t, a964c, tl789c, a!791c, t!792c, cl796t, and c!797t.

[0047] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 27 with at least three amino acid modifications. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 28 with at least five nucleic acid modifications.

[0048] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 30.

[0049] The presently-disclosed subject matter further includes cells comprising the nucleic acid sequences and amino acid sequences disclosed herein. The presently-disclosed subject matter further includes genetically-modified rodents. Embodiments of the presently -disclosed subject matter include methods for assessing a candidate therapeutic that contains trastuzumab, pertuzumab, derivatives thereof, and / or drug conjugates thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0051] FIG. 1 is a graph showing the Number of predicted neoantigens on the human HER2 sequence as compared to the designed HER2Xsequence.

[0052] FIG. 2A-2C depict predicted protein structures of HER2Xon human and mouse HER2. FIG. 2A depicts the mouse HER2 (left), Her2x(middle), and human HER2, with the trastuzumab binding region on the left side of each structure. FIG. 2B depicts the trastuzumab binding region of the mouse HER2-HER2Xoverlay (left) and of the human HER2-HER2Xoverlay (right). FIG. 2C highlights trastuzumab binding from FIG. 2B, including mouse HER2-HER2Xoverlay (left) and of the human HER2-HER2Xoverlay (right).

[0053] FIG. 3 includes the flow cytometry' results analyzing trastuzumab binding and cell surface expression of human and mouse HER2 for mouse HER2, human HER2, and the HER2Xreceptor sequences transduced into EMT6 murine mammary cancer cells.

[0054] FIG. 4A-4B include tumor growth curves of each EMT6 model in athymic nude mice (FIG. 4A) and immunocompetent BALB / c mice (FIG. 4B).

[0055] FIG. 5A-5C include results of flow cytometry- analyses of infiltrating immune cells in tumors extracted from the immunocompetent mice of FIG. 4A-4B.

[0056] FIG. 6A-6C include results of flow cytometry analyses of tumor cell expression of HER2 and trastuzumab binding in tumors extracted from the immunocompetent mice of FIG. 4A-5B

[0057] FIG. 7A-7B shoyv EMT6 HER2X tumors display response and sensitivity' to T-DXd in vitro and in vivo. FIG. 7A includes cell viability curves of EMT6 parental and EMT6HER2X cells treated for 72 hours with various concentrations of T-DXd, FIG. 7B includes tumor growth and survival curves of each EMT6 model in BALB / c mice treated weekly with vehicle or T-DXd.

[0058] FIG. 8 includes tumor grow th curves in C57BL / 6 mice implanted with E0771 murine mammary cancer cells transduced with mouse HER2 and HER2Xsequences.

[0059] FIG. 9A-9B include Growth curves of E0771 HER2X(pink) and mouse HER2 (green) expressing tumors in C57BL / 6 mice treated weekly with vehicle or T-DXd. FIG. 9A includes grouped growth curves. FIG. 9B includes individual growth curves of each mouse, including mouse HER2-expressing control tumors (left) and HER2x-expressing tumors (right).

[0060] FIG. 10A-10B provide schematic depictions of the process for generating a HER2Xmouse.

[0061] FIG. 11 is a Southern blot, providing confirmation of F0 C57CL / 6 mice bearing the transgene and confirmation of HER2X+ N1 mice.

[0062] FIG. 12 includes the flow cytometry results analyzing trastuzumab and pertuzumab binding and cell surface expression of human and mouse HER2 for TerPer receptor sequences transduced into EMT6 murine mammary cancer cells.

[0063] FIG. 13A-13B includes flow- cytometry results analyzing trastuzumab and pertuzumab binding on EMT6 TraPer HER2 expressing cells.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0064] SEQ ID NO: 1 is a trastuzumab binding region of the mouse HER2 amino acid sequence.

[0065] SEQ ID NO: 2 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1

[0066] SEQ ID NO: 3 is a modified trastuzumab binding region of the mouse HER2 amino acid sequence, including S15P, S16P, and SUF amino acid modifications relative to SEQ ID NO: 1

[0067] SEQ ID NO: 4 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0068] SEQ ID NO: 5 is a modified trastuzumab binding region of the mouse HER2 amino acid sequence, including with S15P, S16P, S17F, SIP, E7V, Y38F, and 144A amino acid modifications relative to SEQ ID NO: 1.

[0069] SEQ ID NO: 6 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5

[0070] SEQ ID NO: 7 is a pertuzumab binding region of the mouse HER2 amino acid sequence.

[0071] SEQ ID NO: 8 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7

[0072] SEQ ID NO: 9 is a modified pertuzumab binding region of the mouse HER2 amino acid sequence, including P66L and N67H amino acid modifications relative to SEQ ID NO: 7

[0073] SEQ ID NO: 10 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9

[0074] SEQ ID NO: 11 is the mouse HER2 amino acid sequence, including a trastuzumab binding region (aa580-aa628) and a pertuzumab binding region (aa253-aa343).

[0075] SEQ ID NO: 12 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 , including the trastuzumab binding region (ntl 738-ntl 884) and a pertuzumab binding region (nt757-ntl011).

[0076] SEQ ID NO: 13 is a fragment of the mouse HER2 amino acid sequence, including a trastuzumab binding region (aa328-aa376) and a pertuzumab binding region (aal- aa91).

[0077] SEQ ID NO: 14 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13, including the trastuzumab binding region (nt982-ntl l28) and a pertuzumab binding region (ntl-nt275).

[0078] SEQ ID NO: 15 is a modified fragment of the mouse HER2 amino acid sequence, including P66L, N67H, S342P, S343P, and S344F modifications relative to SEQ ID NO: 13.

[0079] SEQ ID NO: 16 is a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 15

[0080] SEQ ID NO: 17 is a trastuzumab binding region of the rat HER2 amino acid sequence.

[0081] SEQ ID NO: 18 is a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 17

[0082] SEQ ID NO: 19 is a modified trastuzumab binding region of the rat HER2 amino acid sequence, including S15P, S16P, and S17F amino acid modifications relative to SEQ ID NO: 17

[0083] SEQ ID NO: 20 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19

[0084] SEQ ID NO: 21 is a pertuzumab binding region of the rat HER2 amino acid sequence.

[0085] SEQ ID NO: 22 is a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 21

[0086] SEQ ID NO: 23 is a modified pertuzumab binding region of the rat HER2 amino acid sequence, including P66L and N67H amino acid modifications relative to SEQ ID NO: 21

[0087] SEQ ID NO: 24 is a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 23

[0088] SEQ ID NO: 25 is the rat HER2 amino acid sequence, including a trastuzumab binding region (aa583-aa631) and a pertuzumab binding region (aa256-aa346).

[0089] SEQ ID NO: 26 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, including a trastuzumab binding region (nt 1747 -nt 1894) and a pertuzumab binding region (nt766-ntl040).

[0090] SEQ ID NO: 27 is a fragment of the rat HER2 amino acid sequence, including a trastuzumab binding region (aa328-aa376) and a pertuzumab binding region (aal-aa91).

[0091] SEQ ID NO: 28 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27, including a trastuzumab binding region (nt982-ntl 129) and a pertuzumab binding region (ntl-nt275).

[0092] SEQ ID NO: 29 is a modified fragment of the rat HER2 amino acid sequence, including P66L, N67H, S342P, S343P, and S344F modifications relative to SEQ ID NO: 27.

[0093] SEQ ID NO: 30 is a nucleotide sequence encoding the ammo acid sequence of SEQ ID NO: 29.

[0094] SEQ ID NO: 31 is a trastuzumab binding region of the human HER2 amino acid sequence.

[0095] SEQ ID NO: 32 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31

[0096] SEQ ID NO: 33 is a pertuzumab binding region of the human HER2 amino acid sequence.

[0097] SEQ ID NO: 34 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33

[0098] SEQ ID NO: 35 is the human HER2 amino acid sequence, including a trastuzumab binding region (aa579-aa627) and a pertuzumab binding region (aa252-aa342).

[0099] SEQ ID NO: 36 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35, including a trastuzumab binding region (ntl735-ntl881) and a pertuzumab binding region (nt754-ntl026).

[0100] SEQ ID NO: 37 is a fragment of the human HER2 amino acid sequence, including a trastuzumab binding region (aa328-aa376) and a pertuzumab binding region (aal- aa91).

[0101] SEQ ID NO: 38 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, including a trastuzumab binding region (nt982-ntl 128) and a pertuzumab binding region (ntl-nt255).DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0102] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary7skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary7limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0103] The presently-disclosed subject matter includes humanized rodent HER2 sequences. Embodiments of the presently -disclosed subject matter include polypeptides and nucleic acids, cells comprising such nucleic acids and expressing such polypeptides, and genetically -modified rodents. Embodiments of the presently-disclosed subject matter include methods for assessing a candidate therapeutic that contains trastuzumab, pertuzumab, derivatives thereof, and / or drug conjugates thereof.

[0104] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 1 in which 3-7 ammo acids have been modified, wherein the amino acid modifications comprise S15P, S16P, and S17F, and optionally further comprise one or more amino acid modifications selected from the group consisting of SIP, E7V, Y38F, and I44A. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 7 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 17 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S15P, S16P, and S17F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 21 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

[0105] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 9. In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 3 and SEQ ID NO: 9 In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 5 and SEQ ID NO: 9.

[0106] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 23. In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 19 and SEQ ID NO: 23

[0107] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 in which 3-7 amino acids have been modified, wherein the amino acid modifications comprise S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S580P, E586V, Y617F, and 1623A. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 in which 5-9 amino acids have been modified, wherein the amino acid modifications comprise P318L, N319H, S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S580P. E586V. Y617F, and 1623A. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 11 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P318L and N319H.

[0108] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 25 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S597P, S598P, and S599F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 25 in which 3-5 amino acids have been modified, wherein the amino acid modifications comprise P321L, N322H, S597P, S598P, and S599F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 25 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P321L and N322H.

[0109] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 13 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S342P, S343P, and S344F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 13 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

[0110] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 13 in which 5 amino acids have been modified, wherein the amino acid modifications comprise P66L, N67H. S342P. S343P. and S344F. In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 15.

[0111] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 27 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S342P, S343P, and S344F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 1 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

[0112] In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 27 in which 5 amino acids have been modified, wherein the amino acid modifications comprise P66L, N67H. S342P. S343P. and S344F. In some embodiments, a sequence is provided comprising the amino acid sequence of SEQ ID NO: 29.

[0113] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 2 in which 6-13 nucleotides have been modified, wherein the nucleotide modifications comprise t43c, a45c, t46c. t48c. c50t, and c51t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tic, g3c, a20t, al !3t, c! 14t, al30g, and t!31c. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 8 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise 197t, g!98t, a!99c, and c201t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 18 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise t43c, a45c, t46c, c50t, and c51t. In someembodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 22 in which 3 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t, and al99c.

[0114] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 10. In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 4 and SEQ ID NO: 10 In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 6 and SEQ ID NO: 10

[0115] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 24. In some embodiments, a sequence is provided comprising the sequence of SEQ ID NO: 20 and SEQ ID NO: 24

[0116] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 in which 6-13 nucleotides have been modified, wherein the nucleotide modifications comprise t!780c, a!782c, t!783c, tl785c. c!787t, and c!838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of t!738c, gl740c, a!757t, a!850t, cl 85 It, al 867g, and tl 868c. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 in which 10-17 nucleotides have been modified, wherein the nucleotide modifications comprise c953t, g954t, a955c, c957t, t!780c, a!782c, 11783c. tl785c. cl787t, and c!838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tl 738c, gl 740c, al757t, al 850t, cl 8511, al 867g, and t!868c. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 12 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise c953t, g954t, a955c, and c957t.

[0117] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise t!789c, al791c. t!792c, cl796t, and cl797t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise c962t, g963t, a964c, t!789c, a!791c, t!792c, c!796t, andc!797t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 26 in which 3 nucleotide have been modified, wherein the nucleotide modifications comprise c962t, g963t, and a964c.

[0118] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 14 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise c!024t, g!025t. al 026c, and c!028t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 14 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise c!97t, g!98t, a!99c, and c201t.

[0119] In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 14 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise c!97t, gl98t, al 99c, c201t, c!024t, gl025t, al 026c, and c!028t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 16

[0120] In some embodiments of the presently-disclosed subject matter, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 28 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise tl024c, al 026c, tl027c, c!031t, and c!032t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 28 in which 3 nucleotides have been modified, wherein the nucleotide modifications comprise c!97t, g!98t, and a!99c.

[0121] In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 28 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise c!97t, g!98t, a!99c, tl024c, a!026c, tl027c, clO31t, and cl032t. In some embodiments, a sequence is provided comprising the nucleotide sequence of SEQ ID NO: 30

[0122] The presently-disclosed subject matter further includes a vector that comprises any of nucleotide sequences as disclosed herein.

[0123] The presently-disclosed subject matter further includes a cell that comprises any of the nucleotide sequences and / or polypeptide sequences as disclosed herein. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a mouse or rat cell. Insome embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a mammary cancer cell. In some embodiments, the cell is a normal / non-cancer cell.

[0124] The presently-disclosed subject matter further includes a genetically-modified rodent, comprising a cell modified to express any of the polypeptides as disclosed herein. In some embodiments, the polypeptide is expressed in a specific tissue in the rodent. In some embodiments of the genetically -modified rodent, the specific tissue comprises tumor cells. In some embodiments of the genetically-modified rodent, the specific tissue is mammary tissue. In some embodiments, the rodent is a mouse or a rat.

[0125] The presently-disclosed subject matter further includes a genetically-modified rodent, comprising any of the nucleic acids as disclosed herein. In some embodiments, the nucleic acid is knocked into the rodent HER2 / ERBB2 locus. In some embodiments, the nucleic acid expresses a polypeptide in a specific tissue in the rodent. In some embodiments, the specific tissue comprises tumor cells. In some embodiments, the specific tissue is mammary tissue. In some embodiments, the rodent is a mouse or a rat.

[0126] The presently-disclosed subject matter further includes a method of assessing a candidate therapeutic that contains trastuzumab, pertuzumab, a derivative thereof, and / or a drug conjugate thereof. In some embodiments, the method involves contacting the candidate therapeutic to a cell as disclosed herein and assessing the effect of the candidate therapeutic on the cell. In some embodiments of the method, the cell is a rodent cancer cell, and assessing the effect of the candidate therapeutic comprises determining cell viability and / or determining selectivity for HER2. In some embodiments, the rodent cancer cell is a mouse or a rat cancer cell.

[0127] Some embodiments of the method involve administering the candidate therapeutic to a genetically-modified rodent as disclosed herein and assessing the effect of the candidate therapeutic on the rodent. In some embodiments of the method, the rodent comprises tumor cells, and assessing the effect of the candidate therapeutic comprises determining tumor cell viability and / or determining selectivity for HER2. In some embodiments of the method, the candidate therapeutic comprises trastuzumab, pertuzumab, and / or a derivative thereof, conjugated to a therapeutic drug. In some embodiments, the therapeutic drug is a chemotherapeutic of cytotoxic agent useful for treating cancer. In some embodiments, the rodent is a mouse or rat.

[0128] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0130] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0131] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability' and public dissemination of such information.

[0132] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (See, iubmb.qmul.ac.uk / ).

[0133] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0134] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the filing date of this Application.

[0135] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0136] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0137] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%. in some embodiments ±0. 1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0138] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11. 12, 13, and 14 are also disclosed.

[0139] The present application can “comprise” (open ended) or “consist essentially of' the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0140] A “derivative” of trastuzumab or pertuzumab refers to a modified version of trastuzumab or pertuzumab in which one or more amino acid substitutions, deletions, insertions, or chemical modifications have been introduced, provided that the derivative retains the ability to bind to the target receptor with an affinity comparable to or greater thanthat of trastuzumab or pertuzumab. These modifications may be introduced to enhance stability7, improve binding affinity, reduce immunogenicity, or confer other desirable properties while maintaining the antibody’s specificity for the target receptor.

[0141] A “drug conjugate’' of trastuzumab or pertuzumab refers to refers to a complex in which a therapeutic agent, such as a cytotoxic drug or biologically active molecule, is covalently attached to trastuzumab or pertuzumab. The trastuzumab or pertuzumab in the conjugate serves to specifically bind to the target receptor, facilitating targeted delivery7of the therapeutic agent to the desired site of action. The conjugate is designed to retain trastuzumab or pertuzumab’ s specificity7for their target receptors while ensuring that the attached drug remains inactive until released at or within the target cells, thereby enhancing therapeutic efficacy and minimizing off-target effects. As used herein, the term “trastuzumab-drug conjugate” and “pertuzumab-drug conjugate” are inclusive of drug conjugates that make use of a trastuzumab derivative or pertuzumab derivative.

[0142] As used herein, a “modification” is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying such sequences are routine to those of skill in the art.

[0143] The terms “polypeptide fragment” or “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is the same as the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy -terminus of the reference polypeptide, or alternatively both.

[0144] The terms “nucleotide fragment” or “fragment,” when used in reference to a reference nucleotide sequence, refer to a nucleotide sequence in which nucleotides are deleted as compared to the reference nucleotide sequence itself, but where the remaining nucleotide sequence is the same as the corresponding positions in the reference sequence. Such deletions can occur at the 5’ end or the 3’ end of the reference nucleotide sequence, or alternatively both.

[0145] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includesinstances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0146] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.EXAMPLES

[0147] Example 1: Generation of a minimally-edited chimeric HERZ receptor (HER2X).

[0148] A human-rodent chimeric HER2 receptor amino acid sequence was designed and identified as "HER2X,” which includes three (3) amino acid modifications relative to wild type. For mouse, the amino acid sequence is that of SEQ ID NO: 11, including the following amino acid modifications: S594P, S595P, and S596F. For rat, the amino acid sequence is that of SEQ ID NO: 25, including the following amino acid modifications: S597P, S598P, and S599F.

[0149] In the context of creating and testing a chimeric human-rodent receptor for expression in a rodent model, neoantigens are novel peptide sequences generated from the chimeric receptor that the rodent's immune system recognizes as foreign. These neoantigens arise due to differences between the human and rodent components of the receptor. Eliminating or minimizing these neoantigens is useful for ensuring the receptor’s stability, functionality, and accurate modeling of human biology in the rodent system.

[0150] With reference to FIG. 1, using an MHC class I peptide binding prediction server, the HER2Xsequence, with the three (3) amino acid modifications, was found to eliminate all predicted neoantigens of the fully human HER2 sequence on a C57B1 / 6 murine background. These results supported the use of the HER2Xsequence in an immunocompetent rodent model without concern over immunologic rejection or artificial immunologic background activity.

[0151] Example 2: AlphaFold Overlay of the predicted protein structures of HER2Xon human and mouse HERZ.

[0152] Alphafold protein structure prediction was performed to ensure sequence alterations would not cause dramatic protein misfolding. The amino acid sequences of mouse HER2, human HER2, and HER2X were input into the AlphaFold pipeline and PyMol software was used for 3D visualization, analyses, and alignment of the predicted protein structures. FIG. 2A-2C depict predicted protein structures of HER2Xon human and mouse HER2. FIG. 2A depicts the mouse EIER2 (left), Her2x(middle), and human HER2, with the trastuzumab binding region on the left side of each structure. FIG. 2B depicts the trastuzumab binding region of the mouse HER2-HER2Xoverlay (left) and of the human HER2-HER2Xoverlay (right). FIG. 2C highlights trastuzumab binding of FIG. 2B, showing that the trastuzumab binding region of HER2Xmore closely resembles the binding region of human HER2 compared to mouse HER2.

[0153] Modifying the specific three (3) contiguous amino acids to generate the HER2Xreceptor is not predicted to disrupt receptor folding or structure. This is evidenced in FIG. 2A as the general overall structure of the HER2Xreceptor is similar to the mouse HER2 (left) and the human HER2 (right) predicted structures. Although the HER2Xreceptor preserved the general structure shared by both mouse and human HER2, the trastuzumab binding loop of HER2X(middle and bottom panels) more closely resembles that of human HER2 after in silica chimerization.

[0154] Example 3: HER2Xreceptor binds to trastuzumab.

[0155] After Alphafold analysis predicted the expected protein structural changes, the three (3) amino acid modifications were made and expressed in murine mammary cancer cells. Mouse HER2, human HER2, and the HER2Xreceptor sequences were transduced into EMT6 murine mammary' cancer cells, HER2 non-amplified cells at baseline. Flow cytometry' was used to analyze trastuzumab binding ability of the cells using a 680 flurophore-labelled trastuzumab antibody. Cell surface expression of human and mouse HER2 was also analyzed. SKBR3 cells, a human-derived HER2 amplified cell line, was used as a positive control.

[0156] With reference to FIG. 3, an a-mouse HER2 antibody stained mouse HER2 and HER2X -transduced cells, with some minimal staining of EMT6 parental. An a-human HER2 antibody stained only SKBR3 cells (positive control) and human HER2 -transduced cells. Finally, fluorophore-labelled trastuzumab stained SKBR3 cells, and human HER2 and HER2X -transduced cells. It is noted that, while HER2Xcan now bind trastuzumab, it stillbinds to an anti-mouse HER2 antibody because it retains the mouse HER2 backbone sequence.

[0157] Example 4: EMT6 HER2Xtumors avoid growth suppression and immunologic selection of human HER2-expressing tumors in vivo.

[0158] Tumor growth curves were obtained for each EMT6 model in athymic nude mice (FIG. 4A) and immunocompetent BALB / c mice (FIG. 4B). All tumors grow at equal rates in the athymic nude mice, which are largely lacking an adaptive immune system due to deficiency in mature T cells. However, the human HER2 expressing tumors grow significantly slower in immunocompetent mice. Importantly, the HER2Xexpressing tumors grow at the same rate as the controls in the immunocompetent mice and avoid the growth suppression observed in the human HER2 expressing tumors.

[0159] With reference to FIG. 5A-5C, flow cytometry analyses was performed using infiltrating immune cells in tumors extracted from the immunocompetent mice. Tumors were extracted 18 days after inoculation. Human HER2 expressing tumors extracted from immunocompetent mice display significant increases in CD4+ (FIG. 5B) and CD8+ (FIG. 5C) T cell infiltration compared to controls (parental and mouse HER2), suggesting immunologic rejection. However, HER2Xexpressing tumors do not exhibit significant increases in T cell infiltration compared to controls, suggesting the murine adaptive immune system is tolerant of the HER2Xreceptor.

[0160] Example 5: EMT6 HER2Ztumors retain HER2 expression and trastuzumab binding ability in vivo.

[0161] With reference to FIG. 6A-6C, flow cytometry’ analyses was performed using tumor cell expression of HER2 and trastuzumab binding in tumors extracted from the immunocompetent mice of Example 4. Tumors were extracted 18 days after inoculation. As the data reflect, the EMT6 HER2Xtumors retain HER2 expression and trastuzumab binding ability.

[0162] Example 6: EMT6 HER2Xtumors display response and sensitivity to T- DXd in vitro and in vivo.

[0163] Cell viability curv es of EMT6 parental and EMT6 HER2Xcells treated for 72 hours with various concentrations of T-DXd are provided in FIG. 7A. Viability’ was assessed using the CellTiter-Glo Luminescent Cell Viability Assay. Tumor grow th and survival curves of each EMT6 model in BALB / c mice treated weekly with vehicle or T-DXd are provided in FIG. 7B. Treatments were initiated once tumors reached 50-100mm3. T-Dxd initial dose was 950pg followed by 200pg thereafter.

[0164] With reference to FIG. 7A. EMT6 HER2X cells (black) are more sensitive than EMT6 parental cells (grey) to T-DXd treatment in vitro. With reference to FIG. 7B, EMT6 HER2Xexpressing tumors (black) are highly responsive and sensitive to T-DXd in vivo. 60% of mice inoculated with HER2Xexpressing tumors had complete tumor response to T-DXd treatment. This response is exclusively observed in HER2Xtumors and is not seen in the EMT6 parental or mouse HER2-treated tumors.

[0165] Example 7: E0771 HER2Xtumors also exhibit normal growth in C57BL / 6 mice.

[0166] With reference to FIG. 8, mouse HER2 and HER2Xsequences were transduced into E0771 murine mammary cancer cells. HER2 non-amplified cells at baseline, and implanted into C57BL / 6 mice. As the data reflect, E0771 HER2Xtumors exhibit normal grow th in C57BL / 6 mice.

[0167] Example 8: E0771 HER2Xtumors display response to T-DXd treatment in vivo.

[0168] With reference to FIG. 9A-9B, growth curves are provided for E0771 HER2X- and mouse HER2-expressing tumors in C57BL / 6 mice treated weekly with vehicle or T-DXd. Treatments were initiated once tumors reached 50-100mm3. T-Dxd initial dose was 950ug followed by 200ug thereafter. FIG. 9A includes grouped growth curv es. FIG. 9B includes individual growth curves of each mouse, including mouse HER2-expressing control tumors (left) and HER2x-expressing tumors (right). As the data reflect, E0771 HER2X- expressing tumors are more responsive and sensitive to T-DXd treatment than mouse HER2- expressing control tumors in vivo.

[0169] Example 9: Generating a HER2Xmouse.

[0170] FIG. 10A-10B provide schematic depictions of the process for generating a HER2Xmouse. With reference to FIG. 10A. the generation of the HER2Xmouse involves genetic engineering to replace the wild-type mouse HER2 receptor (mouse Erbb2) with the HER2Xreceptor (chimeric Erbb2). With reference to FIG. 10B, this process begins with designing DNA constructs encoding the desired modification, such as the insertion of HER2Xsequences or green fluorescent protein (GFP) markers for tracking. These constructs are introduced into mouse embryos through techniques known in the art. One common method involves incubating the DNA with a virus, which facilitates the delivery of the genetic material into the cells. Another approach utilizes CRISPR-Cas9 technology7, where a HiFi Cas9 protein and guide RNAs specific to the mouse HER2 gene target the precise genomic location for editing. The guide RNAs direct the Cas9 protein to create a double-strand break at the mouse HER2 locus, and the engineered DNA template is integrated into the genome via homology-directed repair.

[0171] After these molecular steps, the edited embryos are transferred into a surrogate mouse via embryo transfer. Following gestation, about 20-60% of the live-bom pups carry the desired genetic edit to express the HER2Xreceptor. These genetically modified mice are then screened and validated to confirm successful incorporation of the humanized receptor sequence.

[0172] With reference to FIG. 11, validation was conducted to confirm N1 derivation from founder Erbb2 SSS^PPF HER2Xmutation. A commercially produced AAV1 and electroporation to introduce a single stranded DNA template that includes the desired HER2Xmutations (3 amino acids) into single cell mouse embryos with CRISPR editing reagents. The results depicted in FIG. 11 serve as confirmation of F0 C57CL / 6 mice bearing the transgene, with no obvious changes in fertility or fecundity7and confirmation of HER2X+ N1 mice.

[0173] The resulting HER2Xmouse have endogenous expression of HER2Xon normal tissues and organs, allowing for uptake of T-DXd and toxicity studies.

[0174] Example 10; Generation of a chimeric HERZ receptor including trastuzumab binding site and pertuzumab binding region (TraPer).

[0175] A human-rodent chimeric HER2 receptor amino acid sequence was designed and identified as “TraPer,’' which includes amino acid modifications relative to wild type in the trastuzumab binding region and in the pertuzumab binding region. For mouse, the aminoacid sequence is that of SEQ ID NO: 11, including the following amino acid modifications in the trastuzumab binding region (aa580-aa628): S594P, S595P, and S596F. and the following amino acid modifications in the pertuzumab binding region (aa253-aa343): P318L and N319H. For rat, the amino acid sequence is that of SEQ ID NO: 25, including the following amino acid modifications in the trastuzumab binding region (aa583-aa631): S597P, S598P, and S599F. and the following amino acid modifications in the pertuzumab binding region (aa256-aa346): P321L and N322H.

[0176] Example 11: TraPer receptor binds to trastuzumab and pertuzumab.

[0177] The TraPer HER2 receptor sequence was transduced into EMT6 murine mammary cancer cells, HER2 non-amplified cells at baseline. Flow cytometry was used to analyze trastuzumab and pertuzumab binding, via fluorophore-labelled trastuzumab antibodies, as well as cell surface expression of human and mouse HER2.

[0178] With reference to FIG. 12, the TraPer HER2 expressing cells are able to bind to both trastuzumab and pertuzumab. It is noted that, in this experiment, the trastuzumab and pertuzumab antibodies were labelled with the same fluorophore and therefore could not be run together in the same panel on the flow cytometer. These data demonstrate the ability of the TraPer receptor to bind to trastuzumab and pertuzumab separately.

[0179] TraPer HER2 also accommodates the binding of trastuzumab and pertuzumab in a noncompetitive fashion. With reference to FIG. 13A-13B, flow cytometry analysis of trastuzumab and pertuzumab binding on EMT6 TraPer HER2 expressing cells is provided. Trastuzumab and pertuzumab were labelled with distinct fluorophores, allowing for analyses of trastuzumab and pertuzumab binding simultaneously. Because the two antibodies were labelled with different fluorophores, these data confirm that both trastuzumab and pertuzumab are able to bind to the TraPer HER2 sequence at the same time.

[0180] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:REFERENCES1 . Taha Zaid, Crupi Mathieu J.F., Alluqmani Nouf, Fareez Faiha, Ng Kristy, Sobh Judy, Lee Emily, Chen Andrew, Thomson Max, Spinelli Marcus M., Ilkow Carolina S., Bell John C., Arulanandam Rozanne, Diallo Jean-Simon (2023) “Syngeneic mouse model of human HER2+ metastatic breast cancer for the evaluation of trastuzumab emtansine combined with oncolytic rhabdovirus.” Front. Immunol, Vol. 14. doi.org / 10.3389 / fimmu.2023. 1181014.2. Gill, Catherine P. and Mark Denham (2020) “Optimized transgene delivery' using third-generation lentiviruses,” Current Protocols in Molecular Biology, 133, el25. doi: 10.1002 / cpmb. 1253. Chen S, Sun S, Moonen D, Lee C, Lee AY, Schaffer DV, He L. CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection. Cell Rep. 2019 Jun 25;27(13):3780-3789.e4. doi: 10.1016 / j.celrep.2019.05.103. PMID: 31242412; PMCID: PMC6693498.4. Bu W, Li Y. In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection. J Vis Exp. 2023 Feb 10;(192). doi: 10.3791 / 64718. PMID: 36847377.5. Lewis Phillips G, Guo J, Kiefer JR, Proctor W, Bumbaca Yadav D, Dybdal N, Shen BQ. Trastuzumab does not bind rat or mouse ErbB2 / neu: implications for selection of non- clinical safety models for trastuzumab-based therapeutics. Breast Cancer Res Treat. 2022 Jan;191(2):303-317. doi: 10.1007 / sl 0549-021-06427-w. Epub 2021 Oct 27. PMID: 34708303; PMCID: PMC8763818.

[0181] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is:

1. A polypeptide, comprising:(a) the amino acid sequence of SEQ ID NO: 1 in which 3-7 amino acids have been modified, wherein the amino acid modifications comprise S15P, S16P, and S17F, and optionally further comprise one or more amino acid modifications selected from the group consisting of SIP, E7V, Y38F. and I44A;(b) the amino acid sequence of SEQ ID NO: 7 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H;(c) the amino acid sequence of SEQ ID NO: 17 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S15P, S16P, and S17F; or(d) the ammo acid sequence of SEQ ID NO: 21 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

2. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

53. The polypeptide of claim 2, and further comprising the amino acid sequence of SEQ ID NO:

94. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

95. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

196. The polypeptide of claim 5, and further comprising the amino acid sequence of SEQ ID NO:

237. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

238. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 11 in which 3-7 amino acids have been modified, wherein the amino acid modifications comprise S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S58OP, E586V, Y617F, and 1623 A.

9. The polypeptide of claim 8, comprising the amino acid sequence of SEQ ID NO: 11 in which 5-9 amino acids have been modified, wherein the amino acid modifications comprise P318L, N319H, S594P, S595P, and S596F, and optionally further comprise one or more amino acid modifications selected from the group consisting of S58OP, E586V, Y617F, and 1623A.

10. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 11 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P318L and N319H.

11. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 25 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S597P, S598P, and S599F.

12. The polypeptide of claim 11, comprising the amino acid sequence of SEQ ID NO: 25 in which 3-5 amino acids have been modified, wherein the amino acid modifications comprise P321L, N322H, S597P, S598P, and S599F.

13. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 25 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P321L and N322H.

14. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 13 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S342P, S343P, and S344F.

15. The polypeptide of claim 14, comprising the amino acid sequence of SEQ ID NO: 13 in which 5 amino acids have been modified, wherein the amino acid modifications comprise P66L, N67H. S342P. S343P, and S344F.

16. The polypeptide of claim 1, and comprising the amino acid sequence of SEQ ID NO: 1517. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 13 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

18. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 27 in which 3 amino acids have been modified, wherein the amino acid modifications comprise S342P, S343P, and S344F.

19. The polypeptide of claim 18, comprising the amino acid sequence of SEQ ID NO: 27 in which 5 amino acids have been modified, wherein the amino acid modifications comprise P66L, N67H, S342P, S343P, and S344F.

20. The polypeptide of claim 1 , and comprising the amino acid sequence of SEQ ID NO: 2921. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 27 in which 2 amino acids have been modified, wherein the amino acid modifications comprise P66L and N67H.

22. A nucleic acid, comprising:(a) the nucleotide sequence of SEQ ID NO: 2 in which 6-13 nucleotides have been modified, wherein the nucleotide modifications comprise t43c, a45c, t46c, t48c, c50t, and c51t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tic, g3c, a20t, al l3t, cl 14t, al30g, and tl31c;(b) the nucleotide sequence of SEQ ID NO: 8 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise 197t, gl98t, al99c, and c201t;(c) the nucleotide sequence of SEQ ID NO: 18 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise t43c, a45c, t46c, c50t, and c51t; or(d) the nucleotide sequence of SEQ ID NO: 22 in which 3 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t. and al99c.

23. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:

624. The nucleic acid of claim 23, and further comprising the nucleotide sequence of SEQ ID NO: 1025. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 1026. The nucleic acid of claim 22. comprising the nucleotide sequence of SEQ ID NO: 2027. The nucleic acid of claim 26, and further comprising the nucleotide sequence of SEQ ID NO: 2428. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 2429. The nucleic acid of claim 22. comprising the nucleotide sequence of SEQ ID NO: 12 in which 6-13 nucleotides have been modified, wherein the nucleotide modifications comprise tl 780c, al782c, tl783c, tl785c, cl787t, and cl838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting of tl738c, gl740c, al757t, al850t, cl851t, al867g, and tl868c.

30. The nucleic acid of claim 29, comprising the nucleotide sequence of SEQ ID NO: 12 in which 10-17 nucleotides have been modified, wherein the nucleotide modifications comprise c953t, g954t, a955c, c957t, tl780c, al782c, tl783c, tl785c, cl787t, and cl838t, and optionally further comprise one or more nucleotide modifications selected from the group consisting oftl738c, gl740c, al757t, al850t, cl851t, al867g, and tl 868c.

31. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 12 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise c953t, g954t. a955c. and c957t.

32. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 26 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise tl789c, al791c, tl792c, cl796t, and cl797t.

33. The nucleic acid of claim 32, comprising the nucleotide sequence of SEQ ID NO: 26 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise c962t, g963t, a964c, tl789c, al791c, tl792c, cl796t, and cl797t.

34. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 26 in which 3 nucleotide have been modified, wherein the nucleotide modifications comprise c962t, g963t, and a964c.

35. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 14 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise cl024t. gl025t, al026c, and cl028t.

36. The nucleic acid of claim 35, comprising the nucleotide sequence of SEQ ID NO: 14 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t. al99c, c201t, cl024t, gl025t, al026c. and cl028t.

37. The nucleic of claim 22, and comprising the nucleotide sequence of SEQ ID NO: 1638. The nucleic acid of claim 22. comprising the nucleotide sequence of SEQ ID NO: 14 in which 4 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t, al99c, and c201t.

39. The nucleic acid of claim 22. comprising the nucleotide sequence of SEQ ID NO: 28 in which 5 nucleotides have been modified, wherein the nucleotide modifications comprise tl 024c, al 026c, tl027c, clO31t, and cl032t.

40. The nucleic acid of claim 39, comprising the nucleotide sequence of SEQ ID NO: 28 in which 8 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t, al99c, tl024c, al 026c, tl027c, cl 03 It, and cl032t.

41. The nucleic acid of claim 22, and comprising the nucleotide sequence of SEQ ID NO: 3042. The nucleic acid of claim 22, comprising the nucleotide sequence of SEQ ID NO: 28 in which 3 nucleotides have been modified, wherein the nucleotide modifications comprise cl97t, gl98t, and al99c.

43. A vector comprising the nucleic acid of any one of claims 22-42.

44. A cell, comprising the polypeptide of any one of claims 1-21.

45. The cell of claim 44, wherein the cell is a rodent cell.

46. The cell of claim 45, wherein the cell is a cancer cell.

47. The cell of claim 46, wherein the cancer cell is a mammary cancer cell.

48. The cell of claim 45, wherein the cell is a normal cell.

49. The cell of claim 45, wherein the rodent cell is a mouse or rat cell.

50. A cell, comprising the nucleic acid of any one of claims 22-42.

51. The cell of claim 50, wherein the cell is a rodent cell.

52. The cell of claim 51, wherein the cell is a cancer cell.

53. The cell of claim 52, wherein the cancer cell is a mammary cancer cell.

54. The cell of claim 51, wherein the cell is a normal cell.

55. The cell of claim 51, wherein the rodent cell is a mouse or rat cell.

56. A genetically-modified rodent, comprising a cell modified to express the polypeptide of any one of claims 1-21.

57. The genetically-modified rodent of claim 56, wherein the polypeptide is expressed in a specific tissue in the rodent.

58. The genetically-modified rodent of claim 56, wherein the specific tissue comprises tumor cells.

59. The genetically-modified rodent of claim 58, wherein the specific tissue is mammary7tissue.

60. The genetically-modified rodent of claim 56, wherein the rodent is a mouse or a rat.

61. A genetically -modified rodent, comprising the nucleic acid of any one of claims 22-42.

62. The genetically -modified rodent of claim 61, wherein the nucleic acid is knocked into the rodent HER2 / ERBB2 locus.

63. The genetically -modified rodent of claim 61. wherein the nucleic acid expresses a polypeptide in a specific tissue in the rodent.

64. The genetically-modified rodent of claim 61. wherein the specific tissue comprises tumor cells.

65. The genetically-modified rodent of claim 64, wherein the specific tissue is mammary7tissue.

66. The genetically-modified rodent of claim 61, wherein the rodent is a mouse or a rat.

67. A method of assessing a candidate therapeutic that contains trastuzumab, pertuzumab, a derivative thereof, and / or a drug conjugate thereof, comprising: contacting the candidate therapeutic to the cell of claim 44 and assessing the effect of the candidate therapeutic on the cell.

68. The method of claim 67, wherein the cell is a rodent cancer cell, and assessing the effect of the candidate therapeutic comprises determining cell viability7and / or determining selectivity for HER2.

69. The method of claim 68, wherein the rodent cancer cell is a mouse or a rat cancer cell.

70. A method of assessing a candidate therapeutic that contains trastuzumab, pertuzumab, a derivative thereof, and / or a drug conjugate thereof, comprising: administering the candidate therapeutic to the genetically-modified rodent of claim 56 and assessing the effect of the candidate therapeutic on the rodent.

71. The method of claim 70, wherein the rodent comprises tumor cells, and assessing the effect of the candidate therapeutic comprises determining tumor cell viability7and / or determining selectivity7for HER2.

72. The method of claim 70, wherein the candidate therapeutic comprises trastuzumab, pertuzumab, and / or a derivative thereof, conjugated to a therapeutic drug.

73. The method of claim 72, wherein the therapeutic drug is a chemotherapeutic of cytotoxic agent useful for treating cancer.

74. The method of claim 70, wherein the rodent is a mouse or rat.

75. A method of assessing a candidate therapeutic that contains trastuzumab, pertuzumab, a derivative thereof, and / or a drug conjugate thereof, comprising:contacting the candidate therapeutic to the cell of claim 50 and assessing the effect of the candidate therapeutic on the cell.

76. The method of claim 75, wherein the cell is a rodent cancer cell, and assessing the effect of the candidate therapeutic comprises determining cell viability and / or determining selectivity for HER2.

77. The method of claim 76, wherein the rodent cancer cell is a mouse or a rat cancer cell.

78. A method of assessing a candidate therapeutic that contains trastuzumab, pertuzumab, a derivative thereof, and / or a drug conjugate thereof, comprising: administering the candidate therapeutic to the genetically-modified rodent of claim 61 and assessing the effect of the candidate therapeutic on the rodent.

79. The method of claim 78, wherein the rodent comprises tumor cells, and assessing the effect of the candidate therapeutic comprises determining tumor cell viability and / or determining selectivity for HER2.

80. The method of claim 78, wherein the candidate therapeutic comprises trastuzumab, pertuzumab, and / or a derivative thereof, conjugated to a therapeutic drug.

81. The method of claim 80, wherein the therapeutic drug is a chemotherapeutic of cytotoxic agent useful for treating cancer.

82. The method of claim 78, wherein the rodent is a mouse or rat.

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