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TitleAdoptive Immunotherapy - Methods and Protocols - B. Ludewig, M. Hoffmann (Humana, 2005) WW
TagsMedical
LanguageEnglish
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Total Pages517
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Page 1

M E T H O D S I N M O L E C U L A R M E D I C I N ETM

Adoptive
Immunotherapy

Edited by

Burkhard Ludewig
Matthias W. Hoffmann

Methods and Protocols

Adoptive
Immunotherapy
Methods and Protocols

Edited by

Burkhard Ludewig
Matthias W. Hoffmann

Page 2

Adoptive Immunotherapy

Page 258

Designing TCR for Cancer Immunotherapy 241

recognition of the peptide/MHC complex. The constant domain is invariant and prima-
rily serves as distance holder to expose the variable domain to the peptide/MHC com-
plex of the antigen-presenting cell (APC) and to enable the association to the CD3
complex and to the coreceptors CD4 or CD8. A disulfide bridge close to the transmem-
brane region covalently links both chains. A transmembrane region with a very short
cytoplasmic tail provides membrane anchoring and accurate association with CD3 sub-
units. Charged pairs of residues inside the hydrophobic core of the TCR/CD3 complex
direct the asymmetric assembly of the subunits (54).

Although mouse and human TCR are of considerable sequence identity with respect
to their constant domains, the usefulness of murine TCR in man may be limited by their
potential immunogenicity. Both the high degree of sequence and structural homology
and the invariant character of mouse constant domains permit their entire replacement
by human C / regions without interfering with TCR function. Humanization beyond
that initial step of domain replacement (“deimmunization”) requires several rounds
of mutational refinements with regard to structural and functional aspects of TCR
biology.

The variable and constant domains of either TCR / chain, each composed of a
twisted anti-parallel sheet, are linked by an extended loop and interact with each other
via a few residues. The interactions of both the juxtaposed variable V and V as well as
the constant domains C and C are complex, and the tight interactions of the C /C
pair in particular govern interchain affinity (23). V /C and V /C interactions are
almost absent and thus facilitate domain replacement.

On a genetic basis, an approach towards humanization took advantage of singular
restriction sites located inside the loop sequence that joins the variable and constant
domains to produce chimerized, so-called partially humanized TCR; in the case of the
mouse TCR chain, a conserved AlwNI-site CAGNNNCTG (wherein N = A/C/G/T),
translationally located aminoterminally to the first C -strand and embedded inside
the conserved PEPA sequence, allows the domain exchange (Fig. 4A).

A full-length human constant C has been isolated irrespective of its subfamily
specificity by coupled RT-PCR/nested PCR from the human Jurkat cell line. The RT-
PCR protocol was identical to the isolation of huC and huC , and has been slightly
modified as described under Subheading 3.2.3. by increasing the annealing tempera-
ture to 55 ± 10°C (RT-PCR: primer pair for_huC TCR: [5'- ATA TCC AGA ACC
CTG ACC CT -3'] / rev_huC TCR [5'- GGG AGC ACA GGC TGT CTT ACA -3']).
For nested PCR, the RT-PCR protocol has been adopted, omitting the RT reaction and
using the high-fidelity Pfu-DNA polymerase whose optimal extension temperature
lies at 72°C (nested PCR: primer pair for_huC TCR_AlwNI [5'- CAG AAC CCA
GAA CCT GCC GTG TAC CAG CTG AGA GAC TCT A -3'] / rev_huC _BamHI
[5'- CG GGAT CCT CAG CTG GAC CAC AGC CGC AGC GTC ATG A -3']). In
order to get a full-length constant region, the reverse primers for RT-PCR have been
located at the 3'-noncoding region close to the stop codons of TCR (as for TCR ).
By introducing two mutations into a primer suited for nested PCR, an AlwNI site has
been designed at the corresponding position in the human as compared to the mouse
residue sequence (Fig. 4A).

Page 259

2
4

2
V

o
ss, K

u
b

all, an
d

T
h

eo
b

ald

2
4

2

Fig. 4. (A) Nucleotide and amino acid sequence alignment of a mouse MDM2(81–88)-specific and a human gp100(280–288)-specific
TCR at the related regions of domain replacement to exemplify the high degree of amino acid homology. Highly conserved amino acids are
indicated in bold. GxG refers to the C-terminal consensus sequence of the CDR3 loop, and IQNPxPAVY to the start of the highly conserved C
domain. Additionally, boxed residues delineate the amino acid sequence of the resulting chimerized molecule. Differing residues are under-
lined. The AlwNI restriction site CAGNNNCTG (italic for the recognized bases, bold for the whole sequence) is located close to the start of the
C domain. Nucleotides that have to be changed in the PCR primer for complementarity to the mouse V region are underlined. (B) Nucleotide
and amino acid sequence alignment of an A2.1-restricted mouse MDM2(81–88)-specific and a human gp100(280–288)-specific TCR . Abbre-
viations are as described in (A). EDL refers to the start of a loop connecting both domains, followed by the highly conserved C domain. The
BstYI restriction site PuGATCPy is located within the EDL consensus sequence. For cloning strategy refer to Subheading 3.3.1.

Page 516

Index 499

antigen specificity regulation, 201
retroviral transduction of humanized

mouse receptors,
heterologous expression in human

T-cells,
selection markers, 246, 247, 251
transduction, selection, and

expansion, 247, 249,
251–253

high-avidity tumor-specific T-cell
generation in transgenic mice,
234

humanization of constructs, 240,
241, 243, 244, 251

materials, 233
murine receptor isolation and

cloning,
cloning of amplification

products, 239, 251
overview, 234–236
RACE polymerase chain

reaction, 236, 237
reverse transcriptase-

polymerase chain reaction,
238, 239

RNA preparation, 236, 249
subcloning into retroviral

expression vectors, 240
rationale, 229, 230
receptor biochemical criteria, 230,

232
single-chain receptor generation,

244–246, 251
retroviral transduction of mouse T-

cells,
adoptive transfer of cells, 207
in vivo studies, 208, 209, 211
materials, 202, 203
mouse T-cell studies,

cytokine staining, 206, 211
infection, 205
spleen cell cultures, 205, 210,

211

transgene expression analysis,
205, 206, 211

overview, 201, 202
retrovirus production,

plasmids, 203, 209, 210
producer cell transfection, 203,

204, 210
tracing of cell, 207, 208

TCR, see T-cell receptor
T-helper cell, see also Regulatory T-

cell,
cytokine secretion assay for isolation

and expansion of tumor-
specific cells,

flow cytometry, 259–263
materials, 258
overview, 257, 258
T-cell isolation from mice

immunized with irradiated
tumor cells, 258, 259, 262, 263

tumor immunity role, 257
TNF, see Tumor necrosis factor
Tolerance,

circumvention, see Cytotoxic T-
lymphocyte; T-cell receptor

hematopoietic stem cell
transplantation and tolerance
induction in nonmyeloablated
mice,

flow cytometry identification of
stem cells, 460, 465–467

immunomagnetic purification of
stem cells,
cell sorting, 463, 464
harvesting, 462, 563
magnetic bead labeling, 463

injection of stem cells, 464, 465
materials, 460, 461
overview, 459, 460
preconditioning of recipient mice,

461, 462
short-term culture of stem cells,

465

Page 517

500 Index

induction by monoclonal antibodies,
306–308

Tumor-associated antigens,
cell-mediated immune response, 137
identification, see Serological

analysis of tumor antigens by
recombinant cDNA expression
cloning

melanoma, 186, 266
multiple myeloma, 128
self antigens in graft-vs-leukemia

effect, 431, 432
Tumor necrosis factor (TNF),

monoclonal antibody clinical
trials, 312–314

Two-dimensional electrophoresis, see
Cytotoxic T-lymphocyte;
Regulatory T-cell

V

VLA-4, monoclonal antibody clinical
trials, 315, 316

W

Western blot,
bifunctional antibodies, 338, 339
epidermal growth factor receptor

kinase activity assessment,
383, 384, 386, 387

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