Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22

Abstract

Cluster of differentiation-22 (CD22) belongs to the sialic acid–binding immunoglobulin (Ig)-like lectin family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B-cell receptor because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia. A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 fragment antigen-binding in complex with the two most membrane-proximal Ig-like domains of CD22 (CD22d6–d7). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target. Cluster of differentiation-22 (CD22) belongs to the sialic acid–binding immunoglobulin (Ig)-like lectin family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B-cell receptor because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia. A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 fragment antigen-binding in complex with the two most membrane-proximal Ig-like domains of CD22 (CD22d6–d7). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target. A rapidly expanding immunotherapy to treat hematological cancer uses ex vivo–modified mature T lymphocytes that are engineered to express chimeric antigen receptors (CARs) specific for a targeted antigen on cancer cells (1Labanieh L. Majzner R.G. Mackall C.L. Programming CAR-T cells to kill cancer.Nat. Biomed. Eng. 2018; 2: 377-391Crossref PubMed Scopus (122) Google Scholar). The engineered CAR is capable of redirecting T cells (chimeric antigen receptor T cell [CAR-T cells]) to specifically target and destroy malignant cells expressing the antigen without major histocompatibility complex restriction (2Harris D.T. Kranz D.M. Adoptive T cell therapies: A comparison of T cell receptors and chimeric antigen receptors.Trends Pharmacol. Sci. 2016; 37: 220-230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Srivastava S. Riddell S.R. Engineering CAR-T cells: Design concepts.Trends Immunol. 2015; 36: 494-502Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In the case of B-cell malignancies, such as B cell–associated leukemias and lymphomas, CD19 is a compelling target for CAR-T cell–based therapies because of its restricted expression to the B-cell lineage, thus reducing off-target global cytotoxic effects. To date, there are two anti-CD19 CAR-T cells approved for use by the US Food and Drug Administration for the treatment of pediatric acute lymphoblastic leukemia and adult diffuse large B-cell lymphoma (4Zhang J. Li J. Ma Q. Yang H. Signorovitch J. Wu E. A review of two regulatory approved anti-CD19 CAR T-cell therapies in diffuse large B-cell lymphoma: Why are indirect treatment comparisons not feasible?.Adv. Ther. 2020; 37: 3040-3058Crossref PubMed Scopus (17) Google Scholar). Although complete tumor regression (70–90% for B-cell acute lymphoblastic leukemia [B-ALL]) can be achieved in a substantial fraction of patients, anti-CD19 CAR-T cell therapy has also suffered resistance in some cases because of the loss of expression of the antigen (5Gardner R. Wu D. Cherian S. Fang M. Hanafi L.-A. Finney O. Smithers H. Jensen M.C. Riddell S.R. Maloney D.G. Turtle C.J. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy.Blood. 2016; 127: 2406-2410Crossref PubMed Scopus (392) Google Scholar, 6Brentjens R.J. Rivière I. Park J.H. Davila M.L. Wang X. Stefanski J. Taylor C. Yeh R. Bartido S. Borquez-Ojeda O. Olszewska M. Bernal Y. Pegram H. Przybylowski M. Hollyman D. et al.Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.Blood. 2011; 118: 4817-4828Crossref PubMed Scopus (928) Google Scholar, 7Brentjens R.J. Davila M.L. Riviere I. Park J. Wang X. Cowell L.G. Bartido S. Stefanski J. Taylor C. Olszewska M. Borquez-Ojeda O. Qu J. Wasielewska T. He Q. Bernal Y. et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci. Transl. Med. 2013; 5177ra38Crossref PubMed Scopus (1390) Google Scholar, 8Maude S.L. Frey N. Shaw P.A. Aplenc R. Barrett D.M. Bunin N.J. Chew A. Gonzalez V.E. Zheng Z. Lacey S.F. Mahnke Y.D. Melenhorst J.J. Rheingold S.R. Shen A. Teachey D.T. et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.N. Engl. J. Med. 2014; 371: 1507-1517Crossref PubMed Scopus (3115) Google Scholar). Cluster of differentiation-22 (CD22) represents an alternative target antigen for CAR-T cells in B-cell malignancies. Indeed, CAR-T cells targeting CD22 have shown potent antineoplastic effects in a phase 1 clinical trial enrolling patients who failed to achieve remission in the CD19 CAR-T cell therapy protocol (9Fry T.J. Shah N.N. Orentas R.J. Stetler-stevenson M. Yuan C.M. Ramakrishna S. Wolters P. Martin S. Delbrook C. Yates B. Shalabi H. Fountaine T.J. Shern J.F. Majzner R.G. Stroncek D.F. et al.CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted.Nat. Med. 2018; 24: 20-28Crossref PubMed Scopus (617) Google Scholar, 10Haso W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). CD22 is a transmembrane glycoprotein expressed on B cells that commonly retain expression in CD19neg tumors (9Fry T.J. Shah N.N. Orentas R.J. Stetler-stevenson M. Yuan C.M. Ramakrishna S. Wolters P. Martin S. Delbrook C. Yates B. Shalabi H. Fountaine T.J. Shern J.F. Majzner R.G. Stroncek D.F. et al.CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted.Nat. Med. 2018; 24: 20-28Crossref PubMed Scopus (617) Google Scholar, 10Haso W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). The canonical function of CD22 is to dampen the activating signal of the B-cell receptor. As part of the sialic acid–binding immunoglobulin (Ig)-like lectin (Siglec) family, the extracellular domain (ECD) of CD22 recognizes glycans terminated in α-2,6-sialic acid, and its binding site is located at the most membrane-distal domain (d1) (11Ereño-Orbea J. Sicard T. Cui H. Mazhab-Jafari M.T. Benlekbir S. Guarné A. Rubinstein J.L. Julien J.P. Molecular basis of human CD22 function and therapeutic targeting.Nat. Commun. 2017; 8: 764Crossref PubMed Scopus (61) Google Scholar). The CD22 ECD is composed of seven Ig-like domains (d1–d7) and contains 12 predicted N-linked glycans (Fig. 1). Binding to sialic acid results in phosphorylation of the CD22 intracellular tyrosine-based inhibitory motifs, subsequent recruitment of tyrosine-protein phosphatase SHP-1, and the dampening of the B-cell response (12Walker J.A. Smith K.G.C. CD22: An inhibitory enigma.Immunology. 2008; 123: 314-325Crossref PubMed Scopus (106) Google Scholar). CD22 itself is covered with N-linked glycans terminated in α-2,6-sialic acid and forms homo-oligomers on the surface of B cells (13Gasparrini F. Feest C. Bruckbauer A. Mattila P.K. Müller J. Nitschke L. Bray D. Batista F.D. Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling.EMBO J. 2015; 35: 1-23PubMed Google Scholar). The tilted conformation adopted by CD22 and the location of the binding site on d1 has been proposed to favor cis-interactions and the formation of these nanoclusters (11Ereño-Orbea J. Sicard T. Cui H. Mazhab-Jafari M.T. Benlekbir S. Guarné A. Rubinstein J.L. Julien J.P. Molecular basis of human CD22 function and therapeutic targeting.Nat. Commun. 2017; 8: 764Crossref PubMed Scopus (61) Google Scholar). The essential components of a CAR include the extracellular antigen-binding domain, usually a single-chain fragment variable, a transmembrane, and hinge region that anchors the receptor on the cell surface and projects the single-chain fragment variable out to the ECD, and intracellular signaling motifs from the T-cell receptor (such as CD3ξ, CD28, 4-1BB, or OX40) that are triggered upon antigen engagement. Thus, CARs are designed to transduce antigen-recognition events into signaling cascades that evokes T-cell effector functions, such as the secretion of cytotoxic factors and proinflammatory cytokines. Previous on the of CAR-T cells the of targeting a membrane-proximal epitope on CD22 ECD W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). to membrane-distal on for the epitope of was at the of the CD22 and domains (Fig. (11Ereño-Orbea J. Sicard T. Cui H. Mazhab-Jafari M.T. Benlekbir S. Guarné A. Rubinstein J.L. Julien J.P. Molecular basis of human CD22 function and therapeutic targeting.Nat. Commun. 2017; 8: 764Crossref PubMed Scopus (61) Google Scholar). the m971 was shown to target a membrane-proximal epitope on and a CAR-T cell the m971 binding achieved effects in of B-ALL in comparison with CAR-T cells of targeting membrane-distal W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). To molecular into antibody recognition that into the of effective CAR-T we and the binding site of m971 on CD22 X. M. Z. Pastan I. Dimitrov D.S. and of human PubMed Scopus Google Scholar). crystal structure the of the most membrane-proximal Ig domains and of CD22 and that m971 at the of CD22 to its effects. Together, our data molecular of antigen recognition underlying the of potent CAR-T cells. To the m971 epitope at we of the membrane-proximal domains and of the extracellular (ECD) of human CD22 in complex with the m971 fragment antigen-binding to 1). The structure was by molecular the m971 as an was derived from its crystal structure in complex with the domain J. Sicard T. Cui H. J. P. Julien basis of by a molecular for antibody antigen-binding 2018; PubMed Scopus Google 1). The of for of the and domains of CD22 to because of crystal interactions and thus in the crystal data and cell of B in a As predicted from the the two CD22 and Ig domains a Ig with in and in (Fig. In the most membrane-proximal domain of the a and two (Fig. S. J. R.J. basis of glycoprotein and Commun. 2016; PubMed Scopus Google Scholar). Thus, the structure in the adopted by membrane-proximal Ig domains the In the CD22 to located in not be because of in this of the two predicted N-linked glycans in was in the and was at in with of the expressed in cells (Fig. The structure that the m971 to the of the CD22 domain (Fig. of the variable domain of the and crystal of m971 that its paratope is for binding its epitope of (Fig. The and and the 1 and of m971 with the and and of CD22 (Fig. The surface on the antigen is and is by the and by the As to in the epitope an N-linked at on (Fig. m971 not include the N-linked on was by binding with a of the site at in a binding to CD22 (Fig. Together, our data that m971 recognizes the ECD to the of CD22 and that N-linked on CD22 not the ability to its To the binding we by in the m971 in and in in to the CD22 (Fig. the m971 paratope contains a in (Fig. on this we to to a or in to the of or at the m971 and CD22 (Fig. with a we that and in comparison with and (Fig. of and in the crystal structure of electrostatic surface of the of and shown as the CD22 domain in the crystal m971 to CD22 is in a interactions and with also with the of m971 The of the surface was with the S. Y. M. S. Li and for with and 2011; PubMed Scopus Google and L. The Molecular and are on a of to The electrostatic surface is with to the and of CD22 from or the or with of The at in the of to a of m971 fragment In the of m971 to the ECD of CD22 was to be in the is with the for the by X. M. Z. Pastan I. Dimitrov D.S. and of human PubMed Scopus Google Scholar). m971 binding to As from our the in the binding of m971 to CD22 by because of a (Fig. Indeed, m971 and CD22 and be with an (Fig. of the for a at in the in an in binding to (Fig. of the for the in its binding to the binding to The in the for results from a (Fig. the of a CAR-T cell the (Fig. the in for CD22 not in cell Together, we that our data of the m971 paratope the to surfaces on the CD22 domain in the (Fig. on and of in the we these as and (Fig. The of the from to the of the structure is located at the the of the CD22 domain and the m971 and is by with the of m971 (Fig. and the is located a by B and of the CD22 domain, with interactions with and (Fig. is located at the region of the CD22 domain, to a and with the of (Fig. and this a the or and of a of to R.J. of in binding PubMed Scopus Google Scholar). The of motifs in CD22 (Fig. and that a in CD22 signal in To the ability of the CD22 membrane-proximal region to we binding of to of to molecular this we CD22 or in the or of m971 with of and the complex formation by (Fig. The of the to an complex formation CD22 and with the of a binding to not the binding of m971 (Fig. as be from the crystal structure because the two are a surface glycoprotein expressed on and mature human B represents a antigen for CAR-T cell immunotherapy against B-ALL J. Q. B. S. Wu T. Z. Z. Y. Qu X. Y. S. Z. Y. Y. Y. et CAR T-cell therapy in refractory or relapsed B acute lymphoblastic PubMed Scopus Google Scholar, H. Wu Z. H. C. Y. D. X. Y. W. Wang C. Y. M. Yang Q. Wang Y. W. CAR-T cells targeting CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic 2020; PubMed Scopus Google Scholar). Here, we have the epitope of m971 by of a of CD22 its two most membrane-proximal domains and m971 structure that m971 to the most membrane-proximal Ig-like domain (d7) in and data also that the epitope of m971 not include the As to the therapeutic antibody for binding to CD22 ECD (11Ereño-Orbea J. Sicard T. Cui H. Mazhab-Jafari M.T. Benlekbir S. Guarné A. Rubinstein J.L. Julien J.P. Molecular basis of human CD22 function and therapeutic targeting.Nat. Commun. 2017; 8: 764Crossref PubMed Scopus (61) Google Scholar). of for binding to glycans on the surface of the glycoprotein is because variable on such as or modified have been in cancer cells N. Y. and of in cancer and and 2015; PubMed Scopus (218) Google Scholar, Y. N. for and and in 2016; PubMed Scopus Google Scholar). it is the of CD22 B and the efficacy of CAR-T cell it has been shown that of cell at the immune the T cell and tumor cell can be an Jensen M.C. Y. Wang J. of chimeric is by target epitope from the cell Immunol. 2008; PubMed Scopus Google Scholar, Y. W. P.A. J.A. D.S. P.K. antigen receptor T cells and potent immune Sci. S. A. 2018; PubMed Scopus Google Scholar, S. S. S. S. in the of chimeric antigen receptors for cancer 8: PubMed Google Scholar). is on the for T-cell receptor signaling P.A. The and Immunol. PubMed Scopus Google Scholar). this the of the target antigen on the tumor cell can an for CAR efficacy L. Q. J. from T cell receptors and chimeric antigen receptors (CARs) on T Immunol. 2020; PubMed Scopus Google Scholar). In of these it is to the of the extracellular of CD22 and two major antigen on B cells for immunotherapy in the ECD of CD19 is composed of a Ig-like domain CD22 with its seven Ig-like domains (d1–d7) (Fig. As targeting the membrane-proximal Ig-like domain on CD22 with CAR-T cells to anti-CD19 CAR-T cells W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). CAR-T cell CAR-T cells of targeting membrane-distal (9Fry T.J. Shah N.N. Orentas R.J. Stetler-stevenson M. Yuan C.M. Ramakrishna S. Wolters P. Martin S. Delbrook C. Yates B. Shalabi H. Fountaine T.J. Shern J.F. Majzner R.G. Stroncek D.F. et al.CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted.Nat. Med. 2018; 24: 20-28Crossref PubMed Scopus (617) Google Scholar, 10Haso W. Lee D.W. Shah N.N. Stetler-Stevenson M. Yuan C.M. Pastan I.H. Dimitrov D.S. Morgan R.A. FitzGerald D.J. Barrett D.M. Wayne A.S. Mackall C.L. Orentas R.J. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.Blood. 2013; 121: 1165-1174Crossref PubMed Scopus (371) Google Scholar). A epitope location was for CAR-T cells against Z. D. Yang H. He Z. X. W. Li L. Wang C. Li Y. Li H. H. H. Q. CAR T cells targeting membrane-proximal epitope of the function against large Scholar). Indeed, CARs targeting the membrane-proximal region on cytotoxic and secretion with membrane-distal As with we out the that membrane-proximal on the targeted CD22 are and thus signal In the membrane-distal on CD22 be in interactions with and CAR Although these the of targeting membrane-proximal extracellular for CAR-T be to these into a that CARs are also be in the of CAR-T cells S. Riddell S.R. Engineering CAR-T cells: Design concepts.Trends Immunol. 2015; 36: 494-502Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, The in the of chimeric antigen receptor T J. Sci. 2020; Scopus Google Scholar). Indeed, CAR-T cells have been shown to be capable of cells target with S. E. Y. M. Park S. CAR T cells to tumor 2017; PubMed Scopus Google Scholar). the human receptor cells with that the of CAR-T cells is with the binding M. A. C. H. T cell by in of the single-chain fragment domain not T cell against target cells Immunol. PubMed Scopus Google Scholar). the of of m971 designed on our we a of binding for m971 In this the in for CD22 of CAR-T cells was not in cell the of effective it is to the in the target is in the of this binding of to domains to the cell surface can be by the of and Indeed, with membrane-proximal such as the glycoprotein J.P. S. J.L. details of recognition by the antibody antigen-recognition and 2008; PubMed Scopus Google and A. A. of as an of the epitope of antibody 2016; Full Text Full Text PDF PubMed Scopus (61) Google to with the that m971 was against a CD22 by X. M. Z. Pastan I. Dimitrov D.S. and of human PubMed Scopus Google Scholar, Z. S. He Y. A. T. D. P. B. O. L. Y. Wang et of by human Sci. S. A. PubMed Scopus Google the of surface to the of the complex the of on are for interactions with head at the surface of the cell (Fig. molecular data that for a of be to with on the cell a in CAR CD22 ECD was and as (11Ereño-Orbea J. Sicard T. Cui H. Mazhab-Jafari M.T. Benlekbir S. Guarné A. Rubinstein J.L. Julien J.P. Molecular basis of human CD22 function and therapeutic targeting.Nat. Commun. 2017; 8: 764Crossref PubMed Scopus (61) Google Scholar). The was from the The was into the W. A and for in PubMed Scopus Google restriction and such that a was at the of the to was the in cells for cells at in a for to the cells by and a a at The was with and with an of to and on a at in to achieve The m971 was by The and of m971 into the W. A and for in PubMed Scopus Google the or by the The and of the in cells at a at a cell of cells and at in a for to and and with a a an and with with 1 and a to the into was a and with a and on a to was with the and of the m971 into cells at a of and at a cell of cells and at in a for to the cells by at for and a a at The was with and with an of to and on a at in to achieve To complex was with the for 1 at The complex was a at in and and and for on with of of to molecular on a at in and was used as a The and the by the and a The was as the in the data as a function of the and the was by the at the signal a that is of its was a from to 1 with an of at 1 in and with in and and The binding of m971 or m971 to CD22 ECD and by the in and with of for at transferred into to baseline for transferred into a of at and to The phase was by a in was the with a in and m971 CAR-T cells by of human T cell was in cells by and was and by the T cell by the the was into by at for the was and T cells into by at at for To CAR the T cells to the cell T cells with cells in with a of for of cells was by the with the to the of with effector cell with target cells and and are for for effector cells and target cells To of the was with to of domain as J. Sicard T. Cui H. J. P. Julien basis of by a molecular for antibody antigen-binding 2018; PubMed Scopus Google and on a The m971 complex was to in of the m971 complex by at in and in of and of an by in the and in data at the at the The of the m971 complex was in W. PubMed Scopus Google Scholar). The structure was by molecular in the variable and domains of from our as and by in P. B. and of PubMed Scopus Google and N. J.J. R. R.J. et A for structure PubMed Scopus Google Scholar). The complex was to 12 in and of the complex by at in and in of and of an by in the and in data at the at the The of the complex was in W. PubMed Scopus Google Scholar). The structure was by molecular in the structure of m971 as a and by in P. B. and of PubMed Scopus Google and N. J.J. R. R.J. et A for structure PubMed Scopus Google Scholar). and are in A. B. J. M. P. the most out of 2013; PubMed Scopus Google Scholar). The crystal have been in the and contains S. Y. M. S. Li and for with and 2011; PubMed Scopus Google Scholar, L. The Molecular Scholar, F. D. of large of 2014; PubMed Scopus Google Scholar). The that have of with the of this in this at the is by the for and Engineering of the of the of the and the of J. X. I. W. Y. D. S. and J. J. D. S. and J. J. T. I. and J. J. T. and J. J. X. T. I. D. H. and Y. F. J. T. and I. J. X. T. I. D. H. Y. and J. X. D. S. and J. J. J. X. T. W. Y. D. S. and J. and was by from the of the the and the J. was by from the of T. S. is a of a

Topics & Concepts