CTNF 18/486,325 CTNF 101190 DETAILED ACTION Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA. Status of the Claims Claims 1, 6, 8-10, 12-14, 16-18, 20, 22-24, 26-27, 31, 34, 36-37, 39 are pending and examined herein. Priority This application, filed 10/13/2023, is a CON of PCT/US2022/024345, filed 05/12/2021, and claims benefit of provisional application 63/174,913 filed on 04/14/2021. This priority is acknowledged and the claims examined herein are treated as having an effective filing date of 04/14/2021. Information Disclosure Statement The Information Disclosure Statement filed 02/20/2024 is acknowledged and have been considered. Claim Rejections - 35 USC § 112 07-30-02 AIA The following is a quotation of 35 U.S.C. 112(b): (b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention. The following is a quotation of 35 U.S.C. 112 (pre-AIA), second paragraph: The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention. Claims 1, 6, 8-10, 12-14, 16-18, 20, 22-24, 26-27, 31, 34, and 36 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claims 1, 6, 8-10, and 12 recites a method of determining the recycling of a plurality of molecules using a two chambered system, wherein “… the determining comprises: introducing the plurality of molecules into a first chamber, wherein: the first chamber is separated from a second chamber by a cell layer, and the first and second chambers comprise an aqueous solution; incubating the plurality of molecules in the first chamber; replacing the aqueous solution in both the first and second chambers; and measuring the amount of the plurality of molecules that is released from the cell layer into the first chamber; wherein the aqueous solution is at physiological pH, and the cell layer comprises Madin-Darby Canine Kidney (MDCK) cells expressing a neonatal Fc receptor (FcRn)” . While the claim recites determining the recycling of a plurality of molecules, it is unclear how the actual amount of molecules present in the first chamber can be used to determine recycling given the information in the claim. This is in part because the relationship between the cell layer and the molecules is unclear, the claim recites “ measuring the amount of the plurality of molecules that is released from the cell layer into the first chamber”, however, there is no recitation describing the mechanism by which the molecules enter the cell layer and are subsequently released back into the first chamber. It is unclear how the molecules in the first chamber are known to be molecules that were recycled specifically, as there are several means through which the molecules could have been released from the cell layer into the first chamber which do not require recycling, e.g. lysis of the cells, leak. Furthermore, Claim 12 recites “ The method of claim 1, comprising incubating the plurality of molecules in the first chamber in the presence of an agent, and determining whether the agent affects the recycling of the plurality of molecules”. However, the recited language of “determining” is indefinite because it is unclear what observations will be made to determine if the agent “affects” the recycling of the molecules, which could be done in several ways. For example, the “determining” could be done by observing whether the rate of recycling is affected, or whether the total number of molecules recycled was affected. Regarding claims 13-14, 16-18, 20, 22-24, claim 13 recites “ A method of determining the tissue penetrance of a plurality of molecules… ” in which step b) teaches “ measuring the amount of the plurality of molecules that is recycled from the first chamber into the cell layer and back to the first chamber ”. Claim 14 similarly recites “ wherein measuring the plurality of molecules that is recycled comprises: ”. These claims are indefinite because it is unclear how it is known that the molecules measured in the first chamber are “recycled” molecules specifically. As discussed in the claim 1 rejection above, there are several means through which the molecules could have been released from the cell layer into the first chamber which do not require recycling. In addition, because claim 13 does not recite replacing the aqueous solution, the molecules measured in the first chamber could simply represent residual molecules that were never transported into the cells of the cell layer, and therefore would not represent recycled molecules. Additionally, claim 24 recites “ The method of any one of claim 13, wherein the tissue penetrance is brain penetrance ”. This claim is indefinite because it is unclear how brain penetrance is determined. For example, it is unclear if the cell layer which separates the first and second is required to be made up of brain cells to measure “brain penetrance”, and whether the ratio of transcytosed to recycled molecules across this layer is a direct measurement of the molecules ability to traffic across the cells which is being deemed “brain penetrance”. In contrast, the “brain penetrance” could be the predicted in vivo ability for molecules to pass through the blood-brain barrier, using the ratio of transcytosed to recycled molecules determined in vitro as a proxy. Claims 26-27, 31, 34, and 36 recites “ A method of identifying an antibody with improved brain penetration capabilities …”. This claim is indefinite because the term “improved” is used in a way that is unclear, as it implies the brain penetration capabilities of an antibody are being compared against another value to determine improvement, though it is unspecified what that value is. For example, the value could be based on a control antibody, a predicted value, or the performance of variant of the antibody being measured or another class of antibody. Furthermore, as discussed with regards to claims 1 and 13-14 above, it is unclear how it is known that the molecules measured in the first chamber are “recycled” molecules specifically. Furthermore, it is unclear in what way the transcytosis/recycling across a cell layer of MDCK cells expressing FcRn indicates brain penetrance. For example, does it predict the in vitro transcytosis/recycling of an antibody across a cell layer composed of brain cells, or does it predict levels found in brain tissue in vivo ? Claim Rejections - 35 USC § 112 07-36 AIA The following is a quotation of 35 U.S.C. 112(d): (d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers. The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph: Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers. 07-36-01 AIA Claim 27 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 27 depends upon claim 26 which recites “ plurality of the antibody molecule”. This limitation of claim 26 establishes that the molecules being measured with regard to transcytosis from the first chamber through the cell layer into the second chamber are antibody molecules. Therefore, claim 27 fails to further limit the subject matter of claim 26, as it recites “ plurality of molecules ” being measured which is broader than existing limitation of “ plurality of the antibody molecule” taught in claim 26 . Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements. Claim Rejections - 35 USC § 112 07-30-01 AIA The following is a quotation of the first paragraph of 35 U.S.C. 112(a): (a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention. The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention. 07-31-01 Claims 13-14, 20, and 22-24 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a written description rejection . Claims 13-14, 20, and 22-24 are directed towards methods of determining the tissue penetrance of a plurality of molecules using and in vitro an assay with two chambers separated by a cell layer comprising cells that express a receptor that mediates molecular transport , which can be used to measure antibody transcytosis through the cell layer into the second chamber, and recycling back into the first chamber. As claimed, these methods lack written description. The specification is silent with respect to the methods as claimed. There are no working examples provided for demonstrating that any type of cell, other than MDCK cells, that expresses a receptor that mediates molecular transport could be used to comprise the cell layer separating the chambers in the assay. Disclosure of drawings or structural chemical formulas/ Actual Reduction to Practice The specification and drawings only provide disclosure of an actual reduction to practice for the use of MDCK cells as the cells comprising the cell layer, not of the claimed invention. The specification lists potential suitable cells to be used in the assays, but this list recites only mammalian and/or endothelial/epithelial cells, and vectors broadly [00131-00133]. Therefore, the claim recites a very broad genus of cells but the specification only describes a single species with no evidence that the genus is contemplated. Level of skill and knowledge in the art/predictability in the art The level of ordinary skill in the art of screening molecules using transcytosis assays is high, as an ordinary artisan in this art needs specialized knowledge of mechanisms of the transport of molecules across various cell types for different molecules. The level of unpredictability in the art is high. In particular, methods measuring transcytosis and recycling to determine tissue penetrance were well known in the art at the time of the invention. At the same time, however, it was not within the skill of the art to predict whether any given cell that expresses a receptor that mediates molecular transport could be used in the methods cell layer to determine tissue penetrance. The likelihood that any cell expressing a receptor that mediates molecular transport could be used in place of MDCK cells, and the ratio of transcytosed to recycled molecules could still be used to determine the tissue penetrance of the plurality of molecules is very low, considering the large amount of variation in structure, function, and transport abilities of such receptors expressed by different cell types. Furthermore, the predictability of using cells other than MDCK cells in an in vitro transcytosis assay to determine tissue penetrance would be low given that the art teaches that for in vitro transcytosis assays the data are often don’t align with the calculated transcytosis capacity of these cells, and may indicate results are influenced unknowingly by paracellular flux (Sade et al. (2014). “A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding”. PLoS One , 9 (4), e96340). Furthermore, the use of cross-species assay systems has been taught in the art as a potential confounding factor , since the binding affinity in proper cell and transport receptor engagement is essential (Sade et al. 2014). In summary, the specification fails to provide adequate written description for the genus of the cell claimed. The specification does not reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention. 07-31-01 Claims 13-14, 16-18, 20 and 24 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a written description rejection . Claims 13-14, 16-18, 20 and 24 are directed towards methods of determining the tissue penetrance of a plurality of molecules using and in vitro an assay with two chambers separated by a cell layer comprising cells that express a receptor that mediates molecular transport, which can be used to measure transcytosis of a plurality of molecules through the cell layer into the second chamber, and recycling back into the first chamber, then determining the tissue penetrance of the plurality of molecules based on the ratio of transcytosed to recycled molecules. As claimed, these methods lack written description. The specification is silent with respect to the methods as claimed. There are no working examples provided for demonstrating that measuring the ratio of transcytosis to recycling for any plurality of molecules, other than 5 anti-BACE1 mAb variants, and determining tissue penetrance based on that ratio. Disclosure of drawings or structural chemical formulas/ Actual Reduction to Practice The specification and drawings only provide disclosure of an actual reduction to practice for the use of 5 anti-BACE1 mAb variants as the plurality of molecules being measured to determine tissue penetrance, not of the claimed invention. While the specification demonstrates the measurement of transcytosis/recycling for other molecules (i.e. humanized IgG1 antibodies, various therapeutic antibodies, and wild type anti-BACE1 mAb and several variants) using this method, only the data and the resulting transcytosis/recycling ratio for the anti-BACE1 mAb variants (when pooled together) was able to be used to determine tissue penetrance. Therefore, the claim recites a very broad genus of molecules but the specification only describes a single suitable species that can be effectively used in the method as claimed. Level of skill and knowledge in the art/predictability in the art The level of ordinary skill in the art of screening molecules using transcytosis assays is high, as an ordinary artisan in this art needs specialized knowledge of mechanisms of the transport of molecules across various cell types for different molecules. The level of unpredictability in the art is high. In particular, methods measuring transcytosis and recycling of molecules to determine tissue penetrance were well known in the art at the time of the invention. At the same time, however, it was not within the skill of the art to predict whether any given molecule could be used in the methods to determine tissue penetrance. The likelihood that any “plurality of molecules” could be used in place of anti-BACE1 mAb variants, and the measured transcytosed to recycled ratio of those molecules could still be used to determine the tissue penetrance of the molecules is very low, considering the large amount of variation in size of molecules that are transported across cell membranes and mechanisms of transport required. For example, many molecules are unable to penetrate brain tissue via the blood-brain barrier due to large size or polarity. Furthermore, the predictability of using any given molecule in an in vitro transcytosis assay to determine its tissue penetrance would be low given that the art teaches that even for the most well-studied molecules in regards to in vitro transcytosis assays such as IgG, the data provides conflicting findings (Ruano-Salguero et al. (2020). “Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn”. Scientific Reports , 10 (1), 3685). In summary, the specification fails to provide adequate written description for the genus of the molecules claimed. The specification does not reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention. Claim Rejections - 35 USC § 112 07-30-01 AIA The following is a quotation of the first paragraph of 35 U.S.C. 112(a): (a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention. The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention. 07-31-02 AIA Claim s 24, 26-27, 31, 34, and 36 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention. Wands factors analysis: The breadth of the claim The claims are broadly directed to a method of determining the tissue penetrance of a plurality molecules, including antibodies (claim 26), by measuring the amount of molecules that are either recycled into and back out of a cell layer (Madin-Darby Canine Kidney cells, MDCK) or transcytosed through the cell layer. Claims 24 and 26 specifically recite that the method is able to determine brain tissue penetrance using the method as claimed. The claim is broad, encompassing potentially many different antibodies to evaluate for improved brain penetration capabilities. Antibodies express large variation in traits like antigen-binding sites, function, and molecular properties between classes, and can also express genetically different allotypes within class. The nature of the invention Claim 24 is directed to a method of determining brain penetrance of a plurality of molecules based on the ratio of transcytosed to recycled molecules measured using an assay with two chambers separated by a cell layer that is comprised of cells that express a receptor that mediates molecular transport , which can be used to measure antibody transcytosis through the cell layer into the second chamber, and recycling back into the first chamber. Claims 26-27, 31, 34, and 36 recite a method of identifying an antibody with improved brain penetration capabilities based on the ratio of transcytosed to recycled molecules measured using an assay with two chambers separated by a layer of MDCK cells expressing FcRn , which can be used to measure antibody transcytosis through the cell layer into the second chamber, and recycling back into the first chamber. The state of the prior art: The prior art teaches the use of transcytosis assays to determine the tissue penetrance of molecules, including brain permeability of antibodies using brain endothelial cells as the cell layer (Ruano-Salguero et al. (2020). “Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn”. Scientific Reports , 10 (1), 3685, (herein referred to as Ruano-Salguero)). The prior art teaches that for a transcytosis assay to generate predictive data for clinical candidate development, the use of a human cellular system and robust, reproducible assay conditions with little inter-assay variability are needed (Sade et al. (2014). “A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding”. PLoS One , 9 (4), e96340). The prior art teaches an in vitro blood-brain barrier model using human brain endothelial cells to study transcytosis, that is also predictive of in vivo rodent CNS antibody exposure for some antibodies (Ribecco-Lutkiewicz et al. (2018). “A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis”. Scientific reports , 8 (1), 1873). The prior art does not teach an in vitro assay using non-brain cells that is predictive of in vivo brain antibody levels. The prior art does not appear to provide any evidence as to in vitro transcytosis/recycling assays using MDCK cells expressing FcRn to determine brain penetrance/permeability as recited in the instant claim. The level of ordinary skill in the art: An ordinary artisan in the area of screening molecules/antibodies for brain penetrating capabilities would have experience in intracellular trafficking assays. Screening of therapeutic antibodies, while complex, is routine in the art. The process of finding new antibodies that have an in vivo pharmacokinetic profile is well known. Additionally, while transcytosis assays are often employed, developing a transcytosis assay using non-brain cells to act as an in vitro model of antibody brain penetrance, as claimed, is generally not well-known or routine, given the complexity of biological systems such as the brain and the mechanisms of intracellular trafficking across the blood-brain barrier. Determining how the in vitro transcytosis and recycling of an antibody mediated by FcRn across non-brain cells will correlate with the levels of the antibody in the brain is not routine. Even with regards to brain endothelial cells, let alone MDCK cells, FcRn’s role in mediating recycling or transcytosis of IgG has remained uncertain. For example, there is no consensus on the role of FcRn in influencing the blood-to-brain transcytosis of IgG across brain endothelial cells. Even less is known about the transcytosis and recycling of antibodies across non-brain cells and its ability to predict the penetrative capacity an antibody levels of a different tissue (brain). Thus, the level of ordinary skill in the art of screening antibodies with improved brain penetration capabilities using transcytosis assays is high, as an ordinary artisan in this art needs specialized knowledge of mechanisms of the transport of antibodies across the blood-brain barrier. The level of predictability in the art: While the ability for some molecules/antibodies to penetrate the brain through the blood-brain barrier and the use of in vitro transcytosis assays using brain endothelial cells is well known, the use of other non-brain cells types in FcRn-dependent transcytosis assays to act as a model to determine brain penetrance within the scope of the claim is not predictable. For instance, the mechanisms regulating IgG shuttling by FcRn are unknown, but are likely tissue and organ-specific (Ruano-Salguero et al. 2020). Therefore, the function of FcRn in brain endothelial cells may not be the same for other endothelium cells in which IgG transcytosis is observed. With regards to claim 24, working examples in the art demonstrating the ability to determine brain penetrance via a transcytosis/recycling assay using a cell layer made of non-brain cell are extremely limited, and the likelihood that any cells that express a receptor that mediates molecular transport could be used for the cell layer are low. The predictability of MDCK cells to an in vitro transcytosis assay to determine brain penetrance would be low given that even for in vitro transcytosis assays using brain endothelial cells, the data often don’t align with the calculated transcytosis capacity of these cells, and may indicate paracellular flux rather than transcytosis (Sade et al. 2014). Because of this, there is not even a consensus on the role of FcRn in influencing the blood-to-brain transcytosis of IgG across brain endothelial cells despite several notable studies (Sade et al. 2014). Furthermore, the use of cross-species assay systems has been taught in the art as a potential confounding factor , since the binding affinity in proper FcRn-IgG engagement is essential (Sade et al. 2014). Another factor that may contribute to the conflicting findings is the influence of antibody concentration on the transcytosis pathway used by endothelial cells. For example, IgG transcytosis across brain endothelial cells has been reported as being concentration dependent (Ruano-Salguero et al. 2020). Experimental methods can influence the amount of endogenous antibody, which could mask the influence of receptors on transcytosis (Ruano-Salguero et al. 2020). The amount of direction provided by inventor and existence of working examples: The specification only shows a relationship between transcytosis/recycling ratio and brain penetrance of molecules using the brain serum ratio in cynomolgus monkeys and the transcytosis/recycling ratio for five anti-BACE1 mAb variants. The specification recites that transcytosis/recycling ratio demonstrated a very strong correlation with the brain serum ratio. While the specification demonstrates this correlation, it is based on only five antibodies, which are all anti-BACE1 mAb variants. Such a relationship could not be thought to apply to all antibodies which have variation in functional and molecular properties. Therefore, the data provided is incongruent with the scope of the claim which recites “antibody” or “antibodies”. The specification does not provide any additional examples or data for other antibodies. Furthermore, even though the correlation recited in the specification was strong, this could have been due to random chance as a byproduct of the small sample size (5 data points). Thus, the specification does not provide sufficient teachings for identifying an antibody with improved brain penetration capabilities with a transcytosis/recycling assay using MDCK cells expressing Fc Rn . The prior art discloses that transcytosis assays using brain endothelial cells are useful in determining the penetrance of antibodies across the blood-brain barrier. The prior art provides no guidance for an in vitro transcytosis/recycling assay using non-brain cells to predict in vivo brain penetrance of antibodies. Therefore, the amount of experimentation required to practice the invention as broadly claimed would not be reasonable. Quantity of Experimentation needed: With regards to claim 24, the amount of experimentation would not be reasonable because it would require a plethora of testing various molecules and cells that comprise the cell layer due to the broad nature of the claim. The “plurality of molecules” can not only be, for example but not limited to, antibodies, antibody fragments, polypeptides, Fc-containing molecules, and ligands, but also a combination of one or more of these molecule types as recited by the specification [0062]. Thus, the genus represented by the scope of the claim is massive. Similarly, the specification provides examples of numerous cell types that could potentially be used to comprise the cell layer [00130], but the scope of the claim includes any cells that express a receptor that mediates molecular transport, which are numerous, and often have different capacities of molecular transport that varies with the molecule type. Furthermore, the scope of the claim would include cells of various species. Therefore, the various molecule and cell type combinations that could be used as recited by the claim is near endless, and the burden of the amount of experimentation that would be needed to determine if the resulting combination from each instance could determine brain penetrance is insurmountable. With regards to claims 26-27, 31, 34, and 36 the amount of experimentation would not be reasonable because it would require determining if the claimed relationship between the transcyctosis/recycling ratio and brain serum ratio for anti-BACE1 mAb variants holds true for other antibodies, which are recited by nature of the claim. Since, as discussed above, it is not routine to use non-brain cells in a transcyctosis/recycling assay to determine in vivo antibody brain penetrance/permeability, and knowing only that the predictive correlation of the instant claim is true for anti-BACE1 mAb variants, it would mean that significant experimentation would be required to determine which other antibodies this correlation may be applicable to. This is because one cannot extrapolate the relationship between the in vitro transcyctosis/recycling ratio determined using MDCK cells and brain serum levels of anti-BACE1 mAb variants, and merely assume the relationship holds true for other antibodies and be used to predict their brain penetrance. There is little guidance (in both the prior art and the specification) with respect to the use of other antibodies. Therefore, the claims are only enabling for anti-BACE1 mAbs. Claim Rejections - 35 USC § 103 07-20-aia AIA The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action: A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made. 07-21-aia AIA Claim s 1, 6, 8-10, 12, and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Chung et al. (2018) . “Development of a label-free FcRn-mediated transcytosis assay for in vitro characterization of FcRn interactions with therapeutic antibodies and Fc-fusion proteins”. Journal of immunological methods , 462 , 101-105, (herein referred to as Chung) in view of Tesar et al. (2006) . “Ligand valency affects transcytosis, recycling and intracellular trafficking mediated by the neonatal Fc receptor”. Traffic , 7 (9), 1127-1142, (herein referred to as Tesar), and Jaramillo et al. (2017) . “Toward in vitro-to-in vivo translation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms”. In MAbs (Vol. 9, No. 5, pp. 781-791). Taylor & Francis, (herein referred to as Jaramillo) Regarding claims 1 and 37 , Chung teaches a method of determining the recycling of a plurality of molecules, wherein the determining comprises: introducing the plurality of molecules into a first chamber (page 103, column 1, 1 st paragraph) , wherein: the first chamber is separated from a second chamber by a cell layer (“MDCK cell monolayer in Transwells”, page 102, column 2, 4 th full paragraph; Fig. 2), and the first and second chambers comprise an aqueous solution (“assay buffer”, page 102, column 2, 5 th full paragraph); incubating the plurality of molecules in the first chamber (page 103, column 1, 1 st paragraph); replacing the aqueous solution in both the first and second chambers (page 103, column 1, 1 st paragraph); wherein the aqueous solution is at physiological pH (“pH 8.0 buffer in the inner and outer chambers “, page 103, column 1, 1 st paragraph), and the cell layer comprises cells that express a receptor that mediates molecular transport which are Madin-Darby Canine Kidney (MDCK) cells expressing a neonatal Fc receptor (FcRn) (page 102, column 1, 3 rd full paragraph; page 102, column 2, 5 th full paragraph). However, Chung does not explicitly teach using their method to determine the recycling of a plurality of molecules. Chung also does not teach measuring the amount of the plurality of molecules that is released from the cell layer into the first chamber as recited in claim 1. Chung also does not teach a detector for detecting the presence of a molecule in the first chamber or second chamber. Tesar teaches the measurement of IgG antibody and Fc ligand transcytosis, recycling and intracellular trafficking with in vitro assays that use transfected MDCK cells expressing rat FcRn (abstract). Tesar teaches the use of MDCK cell monolayers that separates an apical and basolateral reservoir filled with aqueous solution on a plate (page 1139, column 2, 1 st paragraph; page 1140, column 1, 7 th paragraph). Furthermore, Tesar teaches the quantification of recycling by measuring the amount of molecules that is released from the cell layer back into the apical reservoir (page 1134, column 1, 1 st paragraph; page 1140, column 2, 3 rd paragraph). Jaramillo teaches a neonatal FcRn-mediated transcytosis assay in order to investigate monoclonal antibody pharmacokinetics and understand the interplaying clearance mechanisms (abstract). Jaramillo teaches a quantitative transcytosis assay with two chambers filled with solutions of different pH’s that are separated by a monolayer made of MDCK cells transfected with human FcRn (page 789, column 1, 3 rd paragraph). Jaramillo also teaches that samples were taken from both the first and second assay chambers and the amount of the molecules of interest were quantified in these samples using a fluorescence reader system (page 789, column 2, 1 st full paragraph). Furthermore, Jaramillo teaches a detector for detecting the presence of a molecule in the first chamber or second chamber (“Test antibody (3H-labeled, fluorescently labeled or unlabeled)” – page 789, column 2, 1 st full paragraph). It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method taught by Chung to measure the molecules released from the cell layer into the first chamber and to use labeled antibodies, as disclosed in Tesar, and to use a detector to detect the presence of a molecule in the first chamber or second chamber, as disclosed in Jaramillo, in order to determine the amount of antibody recycling back into the first chamber that can occur during FcRn-mediated transcytosis assays using monoclonal antibodies (mAb) (Jaramillo, page 782, column 1, 4 th full paragraph). A skilled artisan would have been motivated to make this modification because it provides an ”… in vitro tool mimicking the entire IgG-FcRn-interaction cycle in living cells, which is a more physiologically-like system” (Jaramillo, page 782, column 1, 3 rd full paragraph), which can be used as a surrogate for antibody recycling in vivo (Jaramillo, page 782, column 2, 1 st paragraph). Such recycling data helps “…to understand the contribution of the FcRn-mediated recycling to mAb PK and highlights that mAb disposition in vivo is a phenomenon likely more complex and more peculiar for each antibody than has been considered so far” (Jaramillo, page 788, column 2, 1 st full paragraph). Measuring the amount of the plurality of molecules that is released from the cell layer into the first chamber allows for antibody recycling to be quantified. A person of ordinary skill would have had a reasonable expectation of success in measuring the amount of molecules that are released from the cell layer into the first chamber, because Jaramillo teaches a quantitative transcytosis assay similar to the one taught by Chung and claim 1 of the application, and shows that a measurement can be taken from the first and second chambers. Therefore, it is merely using a known technique (measuring the molecules in the first chamber) to improve a similar device (transcytosis assay) in order to allow for the predictable improvement of the assay by allowing for measurement of antibody recycling rather than only FcRn interactions with antibodies such as intracellular trafficking. Regarding claim 6, Chung teaches measuring the amount of the plurality of molecules that is released from the cell layer comprising the use of a fluorescence reader system (page 103, column 1, 1 st full paragraph). Regarding claim 8, Chung teaches wherein the plurality of molecules are antibodies (“IgG1 antibodies”, page 102, column 1, 5 th full paragraph). Regarding claim 9, Chung teaches wherein the FcRn selected is human FcRn (page 102, column 1, 4 th full paragraph). Regarding claim 10, Chung teaches measuring the transcytosis of the plurality of molecules across the cell layer, wherein measuring the transcytosis comprises: after the aqueous solution has been replaced in both the first and second chambers, measuring the amount of the plurality of molecules in the second chamber (page 103, column 1, 1 st paragraph). Regarding claim 12, Chung teaches incubating the plurality of molecules in the first chamber in the presence of an agent, and determining whether the agent affects the recycling of the plurality of molecules (“Lucifer Yellow”, page 103, column 1, 1 st paragraph) . 07-21-aia AIA Claim s 13-14, 16-18, 20, 22, 24, 26-27, 31, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Chung, Tesar, and Jaramillo in view of Ruano-Salguero et al. (2020). “ Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn”. Scientific Reports , 10 (1), 3685, (herein referred to as Ruano-Salguero) and Grevys et al. (2018) . “A human endothelial cell-based recycling assay for screening of FcRn targeted molecules”. Nature Communications , 9 (1), 621, (herein referred to as Grevys) . Regarding claims 13-14 and 26-27 , Chung in view of Tesar and Jaramillo teaches an assay to quantify antibody transcytosis and recycling. Chung teaches introducing the plurality of molecules into a first chamber (page 103, column 1, 1 st paragraph), wherein the first chamber is separated from a second chamber by a cell layer (“MDCK cell monolayer in Transwells”, page 102, column 2, 4 th full paragraph; Fig. 2), and the first and second chambers comprise an aqueous solution (“assay buffer”, page 102, column 2, 5 th full paragraph), wherein the aqueous solution is at physiological pH (“pH 8.0 buffer in the inner and outer chambers “, page 103, column 1, 1 st paragraph), and the cell layer comprises cells that express a receptor that mediates molecular transport (page 102, column 1, 3 rd full paragraph; page 102, column 2, 5 th full paragraph). Tesar teaches measuring the amount of the plurality of molecules that is recycled from the first chamber into the cell layer and back to the first chamber (page 1134, column 1, 1 st paragraph; page 1140, column 2, 3 rd paragraph). Chung teaches measuring the amount of the plurality of molecules that is transcytosed from the first chamber to the second chamber (page 103, column 1, first paragraph). Jaramillo also teaches repeating these steps for one or more additional antibodies (Table 2). However, Chung in view of Tesar Jaramillo does not teach determining the brain tissue penetrance of the plurality of antibodies based on the ratio of transcytosed to recycled molecules. Ruano-Salguero teaches measuring the transcytosis of IgG antibodies with native or reduced FcRn engagement across human induced pluripotent stem cell-derived brain endothelial-like cells (abstract). Ruano-Salguero teaches measuring the brain penetrance (“permeability”) of these antibodies to determine the influence of IgG-specific processing occurring within the endothelium of the blood-brain barrier on transcytosis (abstract). Grevys teaches a human endothelial cell-based recycling assay for screening of FcRn targeted molecules (abstract). Grevys teaches that this assay can be used to screen antibodies for relative FcRn-dependent recycling ability to increase the half-lives of IgG and albumin-based therapeutics for greater efficacy (abstract). Grevys also teaches the measurement of the amount of FcRn-mediated trafficking of IgG into the cells (“uptake”, page 8, column 1, 1 st paragraph; Fig. 1 and Fig. 5), as well as the amount that was recycled and released following a 4-hour incubation (“recycling”, page 8, column 1, 1 st paragraph; Fig. 1 and Fig. 5), and also the amount of ligand found intracellularly after the recycling occurred (“residual amount”, page 8, column 1, 1 st paragraph; Fig. 1 and Fig. 5). Grevys further teaches determining a human endothelial cell-based recycling assay score (“HERA score”) by dividing the relative difference in recycling by the relative difference in residual amount (page 12, column 1, 2 nd full paragraph). While Grevys does not explicitly teach examining the ratio of transcytosed to recycled molecules, they do teach examining the ratio of transcytosed molecules remaining in the cell following recycling to the amount of molecules recycled. It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method taught by Chung in view of Tesar and Jaramillo to determine the brain tissue penetrance of the plurality of molecules based on the ratio of transcytosed to recycled molecules. While Ruano-Salguero teaches measuring tissue penetrance, they do not teach measuring the ratio of claim 13. While Grevys teaches a ratio of transcytosed to recycled molecules per say, it is not the exact ratio as outlined in claim 13. However, both Jaramillo and Grevys teach the collection of the data required for determining the ratio of claim 13, but simply do not recite representing the two values as a ratio. Performing such simple calculations with collected data are well known and routinely used by those of ordinary skill in the art. Therefore, it would be “obvious to try” to calculate the ratio of claim 13 as, for example, there are a limited number of ways the data of the amount of molecules going into the cell and being recycled out can be presented. Regarding claims 16-18 , Chung teaches the cell layer comprising Madin-Darby Canine Kidney (MDCK) cells that express a neonatal Fc receptor which mediates molecular transport (FcRn) (page 102, column 1, 3 rd full paragraph; page 102, column 2, 5 th full paragraph). Regarding claim 20 and 31, Chung teaches measuring the amount of the plurality of molecules that is released from the cell layer comprising the use of a fluorescence reader system (page 103, column 1, 1 st full paragraph). Regarding claim 22, Chung teaches wherein the plurality of molecules are antibodies (“IgG1 antibodies”, page 102, column 1, 5 th full paragraph). Regarding claim 24, Ruano-Salguero teaches wherein the tissue penetrance is brain penetrance (abstract). Regarding claim 36, Grevys teaches incubating the plurality of antibody molecules in the first chamber in the presence of an agent, and determining whether the agent affects the ratio of transcytosis to recycling of the antibody (Fig. 8) . 07-22-aia AIA Claim s 23 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Chung in view of Tesar, Jaramillo, Ruano-Salguero, and Grevys as applied to claim s 13-14, 16-18, 20, 22, 24, 26-27, 31, and 36 above, and further in view of Bundhoo et al. (2015). “ Evidence that FcRn mediates the transplacental passage of maternal IgE in the form of IgG anti‐IgE/IgE immune complexes”. Clinical & Experimental Allergy , 45 (6), 1085-1098, (herein referred to as Bundhoo) . The teachings of Chung in view of Tesar, Jaramillo, Ruano-Salguero, and Grevys are incorporated herein. Regarding claims 23 and 34, Chung in view of Tesar Jaramillo, Ruano-Salguero, and Grevys recites all of the limitations of claim 22 and 26 of the application but does not recite wherein the antibody is an anti-IgE antibody, an anti- VEGF antibody, an anti-integrin antibody, an anti-IL-6 antibody, an anti-TNFa antibody, an anti- BACE1 antibody, or an anti-gD antibody. However, Bundhoo teaches the use of MDCK cells stably transfected with human FcRn to study the binding and transcytosis of IgE in the form of IgG anti-IgE/IgE ICs (Summary - Methods). Bundhoo teaches that IgG anti-IgE/IgE immune complexes generated in vitro bound strongly to FcRn-expressing MDCK cells and were transcytosed in an FcRn-dependent manner across placenta cells (Summary – Results). Bundhoo teaches that it is unclear whether cord blood IgE originates in the mother or fetus due to the lack of understanding of the mechanisms involved in the acquisition of materla isotopes other than IgG (Summary – Background). It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method recited in Chung in view of Tesar, Jaramillo, Ruano-Salguero, and Grevys wherein the antibody is an anti-IgE antibody, as disclosed in Bundhoo, as a matter of due to simple substitution. Bundhoo teaches that transcytosis of IgE can be measured with this system because “ FcRn-expressing MDCK cells are a well-established in vitro model system to study IgG transport” - (page 1087, column 1, 1 st full paragraph), and is indeed the same system used by Chung and Jaramillo . A skilled artisan would have been motivated to make this modification because Bundhoo teaches that it is unclear whether cord blood IgE originates in the mother or fetus, and data regarding transcytosis of IgE in this context provides new insight into a pathway for the fetal acquisition of maternal IgE (page 1085, column 1, 1 st paragraph). A person of ordinary skill would have had a reasonable expectation of success in choosing an anti-IgE antibody because Bundhoo teaches that “because hFcRn has strong affinity for human and rabbit IgG, this system was used to evaluate the binding of humanized or rabbit IgG anti-IgEs to hFcRn” . 07-21-aia AIA Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Chung in view of Tesar and Jaramillo as applied to Claims 1, 6, 8-10, 12, and 37 above, and in further view of Kenney et al. (2019) . “Paper-based Transwell assays: an inexpensive alternative to study cellular invasion”. Analyst , 144 (1), 206-211, (herein referred to as Kenney) . The teachings of Chung in view of Tesar and Jaramillo are incorporated herein. Regarding claim 39, Chung in view of Tesar and Jaramillo recites all of the limitations of claim 37 as well as wherein the first and second chambers are components of a trans-well plate, but does not recite that the plate contains 96 wells. However, Kenney teaches paper-based Transwell assays to study cellular invasion that utilizes a standard 96 well plate (abstract; page 206, column 2, 3 rd full paragraph). It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method recited in Chung in view of Jaramillo to use a 96-well transwell plate, as disclosed in Kenney, rather than a 24-well or 6-well plate as disclosed in Chung and Jaramillo, in order to save time and reduce costs. Conclusion For all the reasons discussed above, claims 1, 6, 8-10, 12-14, 16-18, 20, 22-24, 26-27, 31, 34, 36-37, 39 are rejected and therefore no claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to ALEXANDER JOSEPH HOFFMAN whose telephone number is (571)272-9080. The examiner can normally be reached PTA 7:30-5:00 M-F. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy Nguyen can be reached at (571) 272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /ALEXANDER J. HOFFMAN/ Examiner, Art Unit 1677 /BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 May 11, 2026 Application/Control Number: 18/486,325 Page 2 Art Unit: 1677 Application/Control Number: 18/486,325 Page 3 Art Unit: 1677 Application/Control Number: 18/486,325 Page 4 Art Unit: 1677 Application/Control Number: 18/486,325 Page 5 Art Unit: 1677 Application/Control Number: 18/486,325 Page 6 Art Unit: 1677 Application/Control Number: 18/486,325 Page 7 Art Unit: 1677 Application/Control Number: 18/486,325 Page 8 Art Unit: 1677 Application/Control Number: 18/486,325 Page 9 Art Unit: 1677 Application/Control Number: 18/486,325 Page 10 Art Unit: 1677 Application/Control Number: 18/486,325 Page 11 Art Unit: 1677 Application/Control Number: 18/486,325 Page 12 Art Unit: 1677 Application/Control Number: 18/486,325 Page 13 Art Unit: 1677 Application/Control Number: 18/486,325 Page 14 Art Unit: 1677 Application/Control Number: 18/486,325 Page 15 Art Unit: 1677 Application/Control Number: 18/486,325 Page 16 Art Unit: 1677 Application/Control Number: 18/486,325 Page 17 Art Unit: 1677 Application/Control Number: 18/486,325 Page 18 Art Unit: 1677 Application/Control Number: 18/486,325 Page 19 Art Unit: 1677 Application/Control Number: 18/486,325 Page 20 Art Unit: 1677 Application/Control Number: 18/486,325 Page 21 Art Unit: 1677 Application/Control Number: 18/486,325 Page 22 Art Unit: 1677 Application/Control Number: 18/486,325 Page 23 Art Unit: 1677 Application/Control Number: 18/486,325 Page 24 Art Unit: 1677 Application/Control Number: 18/486,325 Page 25 Art Unit: 1677 Application/Control Number: 18/486,325 Page 26 Art Unit: 1677 Application/Control Number: 18/486,325 Page 27 Art Unit: 1677 Application/Control Number: 18/486,325 Page 28 Art Unit: 1677
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